Perm1 enhances Nrf2-driven antioxidant defense through Keap1 oxidation during myocardial ischemia/reperfusion injury
Shin-ichi Oka, Chun-Yang Huang, Masato Matsushita, Allen Sam Titus, Yasuki Nakada, Risa Mukai, Samta Veera, Youssef Mourad, Ghassan Yehia, Peter Romanienko, Yimin Tian, Peiyong Zhai, Junichi Sadoshima
Shin-ichi Oka, Chun-Yang Huang, Masato Matsushita, Allen Sam Titus, Yasuki Nakada, Risa Mukai, Samta Veera, Youssef Mourad, Ghassan Yehia, Peter Romanienko, Yimin Tian, Peiyong Zhai, Junichi Sadoshima
View: Text | PDF
Research Article Cardiology Cell biology

Perm1 enhances Nrf2-driven antioxidant defense through Keap1 oxidation during myocardial ischemia/reperfusion injury

Abstract

Ischemia/reperfusion (IR) enhances oxidative stress, leading to myocardial injury. Although Perm1 promotes cytoprotective mechanisms, the underlying mechanisms are poorly understood. Cysteine oxidation of Keap1 alleviates Cul3-mediated ubiquitination/degradation of Nrf2 and promotes antioxidant transcription. Here we show that Perm1 activates Nrf2 through cysteine oxidation of Keap1 and stabilization of Nrf2. Endogenous Perm1 was downregulated during IR, whereas the rescue of Perm1 reduced IR injury. Downregulation of Perm1 exacerbated oxidative stress, whereas upregulation of Perm1 alleviated it, accompanied by downregulation and upregulation of Nrf2-regulated antioxidant genes, respectively. Perm1 promoted oxidation of cysteine residues in Keap1, possibly through thiol-disulfide exchange reactions, which decreases Keap1-Nrf2 interaction and inhibits Cul3-mediated degradation of Nrf2. We identified Cys121 and Cys746 in Perm1 as critical for Keap1 oxidation and cardioprotection. Thus, Perm1 induces cysteine oxidation of Keap1, thereby conferring myocardial resistance to IR injury by inducing Nrf2 stabilization and transcriptional activation of antioxidant genes.

Authors

Shin-ichi Oka, Chun-Yang Huang, Masato Matsushita, Allen Sam Titus, Yasuki Nakada, Risa Mukai, Samta Veera, Youssef Mourad, Ghassan Yehia, Peter Romanienko, Yimin Tian, Peiyong Zhai, Junichi Sadoshima

×

Figure 5

Perm1 interacts with Keap1.

Options: View larger image (or click on image) Download as PowerPoint
Perm1 interacts with Keap1. (A and B) Perm1 overexpression promoted nucl...
(A and B) Perm1 overexpression promoted nuclear translocation of Nrf2 in cultured cardiomyocytes. Cardiomyocytes were transduced with Ad-Perm1. The levels of indicated proteins were examined in the cytosolic and nuclear fractions. Nuclear translocation of Nrf2 was assessed by immunostaining. (C) Perm1 stimulated reporter gene activity driven by the antioxidant response element (ARE). Statistical significance was determined by Student’s t test (B and C). n = 10–15 (C). *P < 0.05. (D–F) Perm1 binds to Keap1. (D) Coimmunoprecipitation assays were performed with anti-Perm1 antibody using cultured cardiomyocytes. SE, Short exposure; LE, Long exposure. (E) Adenovirus vector carrying Flag-Perm1 was transduced into cardiomyocytes. Flag-Perm1 was immunoprecipitated with anti-Flag antibody. (F) Perm1 bound to Keap1 in vitro. GST-pulldown assays were performed with GST-Perm1 and recombinant Keap1. (G) Perm1 inhibited the binding of Keap1 to Nrf2. Keap1 was immunoprecipitated from cardiomyocytes with Perm1 overexpression. (H) Perm1 did not inhibit the binding of Keap1 to Cul3. (I and J) Loss of Perm1 enhanced the binding of Keap1 and Nrf2. Cultured cardiomyocytes transfected with either siControl or siPerm1 were subjected to sIR. After 30 minutes of hypoxia and 1 hour of reoxygenation, coimmunoprecipitation assays (I) and PLA assays (J) were performed with anti-Nrf2, anti-Keap1, and control IgG antibodies. Statistical significance was determined by ANOVA (G) and Student’s t test (I and J). n = 5 (G), 3 (I) and 12-14 (J). *P < 0.05. (A, D, E, F and H). Representative data are shown from 2 independent experiments. Scale bars: 10 μm (B) and 20 μm (J).