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

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.

Exosome-inflammasome crosstalk and their roles in inflammatory responses

“…These PRRs recognize different PAMPs and DAMPs. Among them, NLRP3 is most frequently investigated and has the most diverse activators 3 , 6 , 30 , 40 , 41 . Unlike other PRRs, NLRP3 and pyrin can be activated without a direct contact with their activators.…”

A Novel Compound, Tanshinol Borneol Ester, Ameliorates Pressure Overload-Induced Cardiac Hypertrophy by Inhibiting Oxidative Stress via the mTOR/β-TrCP/NRF2 Pathway

“…Nuclear factor erythroid 2-related factor 2 (NRF2), a transcription factor, reportedly protects against various oxidative stress-induced diseases and has been suggested as a promising therapeutic target for cardiac oxidative damage (Tian et al, 2021a). Increasing evidence has shown that NRF2 activity is limited in a normal cellular microenvironment (Cheng et al, 2021). However, under constant oxidative conditions, NRF2 released in the cytoplasm from inactivated NRF2-Kelch-like ECH associated protein 1 (Keap1) complex promotes the expression of gene subsets correlated with antioxidant responses such as genes encoding heme oxygenase-1 (HO-1), oxidoreductase 1 (NQO1), and other antioxidant proteins through binding to the antioxidant response elements (ARE) sequences (Nezu and Suzuki, 2021).…”

Interleukin 32 participates in cardiomyocyte‑induced oxidative stress, inflammation and apoptosis during hypoxia/reoxygenation via the NOD2/NOX2/MAPK signaling pathway

“…Given that IL-32 has been implicated in Crohn's disease (18) and rheumatoid arthritis, both of which are associated with inflammation, the present study hypothesized that this cytokine may play a primary role in the development of MIRI. Human cardiomyocytes were used to establish an in vitro model to mimic MIRI by hypoxia and reoxygenation (H/R) (19). The present study investigated the effects of IL-32 on inflammation, oxidative stress and apoptosis in H/R-induced cells.…”

“…Human cardiomyocytes were used to establish an in vitro model to mimic MIRI by hypoxia and reoxygenation (H/R) ( 19 ). The present study investigated the effects of IL-32 on inflammation, oxidative stress and apoptosis in H/R-induced cells.…”