Covalent modification of Keap1 at Cys77 and Cys434 by pubescenoside a suppresses oxidative stress-induced NLRP3 inflammasome activation in myocardial ischemia-reperfusion injury.
Covalent modification of Keap1 at Cys77 and Cys434 by pubescenoside a suppresses oxidative stress-induced NLRP3 inflammasome activation in myocardial ischemia-reperfusion injury.
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毛皂苷 a 对 Keap1 Cys77 和 Cys434 的共价修饰抑制心肌缺血再灌注损伤中氧化应激诱导的 NLRP3 炎性体激活
作者：
Cheng Y;Cheng L;Gao X;Chen S;Wu P;Wang C;Liu Z
背景与目的：Kelch ECH相关蛋白1(Kelch ECH-Associating Protein 1，Keap1)是E3泛素连接酶的重要伴侣蛋白。Keap1中关键的反应性半胱氨酸残基的修饰影响Keap1与其底物核因子红系2相关因子2(Nrf2)之间的相互作用，从而调节氧化应激和NLPR3炎性小体的激活，这是心肌缺血再灌注损伤(MI/RI)的重要因素。毛冬青总皂苷A(Pubescenoside A，PBA)是毛冬青中的一种活性物质，具有抗血栓和抗炎作用。然而，PBA对MI/RI的影响仍不清楚。在本研究中，我们旨在确定PBA是否能保护心脏免受MI/RI的影响，并阐明PBA的直接靶点和潜在机制。方法：采用结扎左前降支(LAD)诱导的MI/RI小鼠模型和缺氧缺糖再灌流(OGD/R)模型，观察PBA的心肌保护作用。通过下拉实验、免疫共沉淀(Co-IP)实验、LC/MS/MS、等温量热(ITC)实验和共价对接等方法对PBA的靶标进行了鉴定。结果：PBA对体外培养的心肌细胞OGD/R和体内LAD诱导的MI/RI均有保护作用。PBA抑制NLRP3炎症激活，诱导Nrf2信号通路。有趣的是，PBA通过选择性地与Keap1保守的半胱氨酸残基、BTB结构域的半胱氨酸77(Cys77)和Keap1的Kelch结构域的半胱氨酸434(Cys434)结合来靶向Keap1，随后抑制Nrf2的泛素化并激活抗氧化酶。此外，Keap1的半胱氨酸被PBA激活的程度如下：Cys77>；Cys434>；Cys23>；Cys38>；Cys226>；Cys273，进一步阐明了Keap1对半胱氨酸的敏感性。结论：PBA可能是一种新的Nrf2激活剂，与Keap1的两个关键区共价结合，具有抗缺血再灌注损伤的心肌保护作用。
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.
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DOI：
10.1107/s1744309104032506
发表时间：
2005-01-01
影响因子：
0.9
作者：
Padmanabhan, B;Scharlock, M;Yokoyama, S
通讯作者：
Yokoyama, S
影响因子：
3.7
作者：
Kim S;Lee HG;Park SA;Kundu JK;Keum YS;Cha YN;Na HK;Surh YJ
通讯作者：
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DOI：
10.1124/jpet.109.160465
发表时间：
2010-01-01
影响因子：
3.5
作者：
He, Xiaoqing;Ma, Qiang
通讯作者：
Ma, Qiang
影响因子：
5.1
作者：
Jiang, ZH;Wang, JR;Liu, L
通讯作者：
Liu, L
影响因子：
3.9
作者：
Dinkova-Kostova AT;Kostov RV;Canning P
通讯作者：
Canning P