Theranostics 2021; 11(2):861-877. doi:10.7150/thno.48436 This issue

Research Paper

Covalent modification of Keap1 at Cys77 and Cys434 by pubescenoside a suppresses oxidative stress-induced NLRP3 inflammasome activation in myocardial ischemia-reperfusion injury

Yuanyuan Cheng1,#, Liangkai Cheng1,#, Xiang Gao2, Sixuan Chen1, Peng Wu1, Caiyan Wang1,✉, Zhongqiu Liu1,✉

1. Guangdong Key Laboratory for Translational Cancer Research of Chinese Medicine, Joint Laboratory for Translational Cancer Research of Chinese Medicine of the Ministry of Education of the People's Republic of China, International Institute for Translational Chinese Medicine, School of Pharmaceutical Sciences, Guangzhou Univ Chinese Med, Guangzhou, Guangdong, 510006, China
2. School of Pharmaceutical Sciences, Xiamen University, Xiamen, Fujian, 361102, China
# Yuanyuan Cheng and Liangkai Cheng contribute to the work equally.

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Citation:
Cheng Y, Cheng L, Gao X, Chen S, Wu P, Wang C, Liu Z. Covalent modification of Keap1 at Cys77 and Cys434 by pubescenoside a suppresses oxidative stress-induced NLRP3 inflammasome activation in myocardial ischemia-reperfusion injury. Theranostics 2021; 11(2):861-877. doi:10.7150/thno.48436. Available from https://www.thno.org/v11p0861.htm

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Abstract

Graphic abstract

Background and Purpose: Kelch ECH-associating protein 1 (Keap1) is a crucial chaperonin for E3 ubiquitin ligases. Modification of the key reactive cysteine residues in Keap1 affects the interaction between Keap1 and its substrate nuclear factor erythroid 2-related factor 2 (Nrf2), subsequently regulating oxidative stress and NLPR3 inflammasome activation, which are important factors for myocardial ischemia-reperfusion injury (MI/RI). Pubescenoside A (PBA), an active compound from Ilex pubescens, has antithrombotic and anti-inflammatory effects. However, the effect of PBA on MI/RI is still unknown. In the present study, we aimed to determine whether PBA can protect the heart against MI/RI and clarify the direct target and the underlying mechanism of PBA.

Methods: The left anterior descending artery (LAD) ligation-induced MI/RI mice model or oxygen and glucose deprivation/reperfusion (OGD/R) were used to evaluate the cardioprotective effect of PBA. Pull-down assays, co-immunoprecipitation (Co-IP) assays, LC/MS/MS, isothermal calorimetry (ITC) experiments and covalent docking were used to identify the target of PBA.

Results: PBA protected cardiomyocytes against OGD/R in vitro and LAD-induced MI/RI in vivo. PBA suppressed NLRP3 inflammation activation and induced the Nrf2 signaling pathway. Interestingly, PBA targeted Keap1 by selectively covalently binding to conserved cysteine residues, cysteine 77 (Cys77) in the BTB domain and cysteine 434 (Cys434) in the Kelch domain of Keap1, subsequently inhibiting ubiquitination of Nrf2 and activating antioxidant enzymes. Additionally, the cysteines of Keap1 has different degree of activation by PBA as follows: Cys77 > Cys434 > Cys23 > Cys38 > Cys226 > Cys273, which further elucidates the cysteine sensitivity of Keap1.

Conclusions: Our results indicated that PBA might be a new Nrf2 activator that covalently binds to two critical domains of Keap1, and shows cardioprotective activities against ischemia-reperfusion injury.

Keywords: pubescenoside A, myocardial ischemia-reperfusion injury, Kelch ECH-associating protein 1, nuclear factor erythroid 2-related factor 2, covalent modification