CISD2 的抑制通过铁蛋白自噬介导的铁蛋白周转和 p62–Keap1–NRF2 通路的调节促进铁死亡,Cellular & Molecular Biology Letters

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CISD2 的抑制通过铁蛋白自噬介导的铁蛋白周转和 p62–Keap1–NRF2 通路的调节促进铁死亡
Cellular & Molecular Biology Letters ( IF 9.2 ) Pub Date : 2022-09-30 , DOI: 10.1186/s11658-022-00383-z
Yanchun Li _{1,

2} , Bing Xu ₃ , Xueying Ren ₄ , Luyang Wang ₂ , Yaqing Xu ₂ , Yefeng Zhao ₂ , Chen Yang ₂ , Chen Yuan ₂ , Huanjuan Li ₂ , Xiangmin Tong ₂ , Ying Wang ₁ , Jing Du ₂

Affiliation

CDGSH 铁硫结构域 2 (CISD2) 是一种具有 [2Fe-2S] 簇的铁硫蛋白，对于细胞增殖和铁稳态至关重要。已证明 CISD2 的异常表达与多种癌症的进展相关。然而，CISD2 调节肿瘤发生的潜在机制仍不清楚。采用生物信息学策略研究 CISD2 的蛋白质相互作用网络和功能注释。功能实验中，采用CCK-8试剂盒测定细胞活力。分别通过 DCF-DA、RPA、C11-BODIPY 和组织蛋白酶 B 染色测定细胞活性氧 (ROS)、细胞内游离铁、脂质过氧化物和溶酶体活性的水平。使用 GSH 测定试剂盒测定谷胱甘肽 (GSH) 含量。我们发现 CISD2 的敲除显着加速了 Erastin 诱导的铁死亡细胞死亡，并伴有过量的脂质过氧化、GSH 耗尽和铁积累，而 CISD2 的过度表达则阻碍了对 Erastin 的敏感性。通过共聚焦显微镜和蛋白质印迹进行的进一步测定表明，CISD2 敲低显着增强了溶酶体活性，并在 Erastin 暴露下激活了铁蛋白自噬。溶酶体功能的药理学抑制可以抑制铁蛋白重链（FTH）的降解，并减弱铁死亡的表型，例如加速铁积累和脂质过氧化。值得注意的是，我们发现 Erastin 诱导的核因子红细胞 2 相关因子 2 (NRF2) 的代偿性升高可以在 CISD2 耗竭细胞中消除。从机械角度来看，CISD2 敲低促进了自噬接头 p62 的降解，并导致 Keap1 与 NRF2 的结合亲和力增加，从而导致 NRF2 泛素化增加和随后的降解。NRF2 的强制表达在体外和体内逆转了 shCISD2 细胞对铁死亡的敏感性。相反，Keap1的强制表达加剧了NRF2的降解，减少了FTH和血红素加氧酶1（HO-1）的转录表达，增加了氧化损伤，从而进一步促进了铁死亡。综上所述，我们目前的结果说明了 shCISD2 介导的铁死亡涉及两种平行机制。一是shCISD2通过铁蛋白自噬依赖性铁蛋白周转来增强游离铁的积累；另一个是 CISD2 缺失会导致 p62-Keap1-NRF2 通路受到抑制，从而导致氧化应激和铁死亡。

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Inhibition of CISD2 promotes ferroptosis through ferritinophagy-mediated ferritin turnover and regulation of p62–Keap1–NRF2 pathway

CDGSH iron sulfur domain 2 (CISD2) is an iron–sulfur protein with a [2Fe–2S] cluster, which is critical for cell proliferation and iron homeostasis. It has been demonstrated that aberrant expression of CISD2 is associated with the progression of multiple cancers. However, the underlying mechanism of CISD2 in regulating tumorigenesis remains obscure. Bioinformatics strategies were used to investigate the protein interaction network and functional annotation of CISD2. In the functional experiment, cell viability was measured by CCK-8 kit. The levels of cellular reactive oxygen species (ROS), intracellular free iron, lipid peroxides, and lysosomal activity were determined by DCF-DA, RPA, C11-BODIPY, and cathepsin B staining, respectively. The glutathione (GSH) content was determined using a GSH assay kit. We showed that knockdown of CISD2 significantly accelerated the Erastin-induced ferroptotic cell death with excess lipid peroxidation, GSH exhaustion, and iron accumulation, while overexpression of CISD2 hindered the sensitivity to Erastin. Further assays via confocal microscopy and western blot exhibited that CISD2 knockdown markedly enhanced the lysosomal activity, and activated ferritinophagy under the exposure of Erastin. Pharmacological inhibition of lysosomal function could inhibit the degradation of ferritin heavy chain (FTH), and attenuate the phenotypes of ferroptosis, such as accelerated iron accumulation and lipid peroxidation. Notably, we found that Erastin-induced compensatory elevation of nuclear factor erythroid 2-related factor 2 (NRF2) could be eliminated in CISD2 depletion cells. Mechanically, CISD2 knockdown promoted the degradation of autophagy adaptor p62 and resulted in an increased binding affinity of Keap1 with NRF2, thus leading to the increased ubiquitination and subsequent degradation of NRF2. Enforced expression of NRF2 reversed the sensitivity of shCISD2 cells to ferroptosis both in vitro and in vivo. Conversely, enforced expression of Keap1 exacerbated the degradation of NRF2, reduced the transcriptional expression of FTH and heme oxygenase 1 (HO-1), increased the oxidative damage, and thus further facilitated ferroptosis. Taken together, our current results illustrated two parallel mechanisms involved in the shCISD2-mediated ferroptosis. One was that shCISD2 enhanced the accumulation of free iron via ferritinophagy-dependent ferritin turnover; the other was that CISD2 depletion induced the inhibition of the p62–Keap1–NRF2 pathway, which resulted in oxidative stress and ferroptosis.

更新日期：2022-09-30

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