Department of Central Laboratory, Affiliated Hangzhou First People's Hospital, Zhejiang University School of Medicine, Hangzhou, 310006, Zhejiang, China.
Department of Clinical Laboratory, Laboratory Medicine Center, Zhejiang Provincial People's Hospital (Affiliated People's Hospital, Hangzhou Medical College), Hangzhou, 310014, Zhejiang, China.
Cell Mol Biol Lett. 2022 Sep 30;27(1):81. doi: 10.1186/s11658-022-00383-z.
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.
CDGSH 铁硫域 2（CISD2）是一种具有[2Fe-2S]簇的铁硫蛋白，对细胞增殖和铁稳态至关重要。已经证明，CISD2 的异常表达与多种癌症的进展有关。然而，CISD2 调节肿瘤发生的潜在机制尚不清楚。
使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。在功能实验中，通过 CCK-8 试剂盒测量细胞活力。通过 DCF-DA、RPA、C11-BODIPY 和组织蛋白酶 B 染色分别测定细胞内活性氧（ROS）、细胞内游离铁、脂质过氧化物和溶酶体活性。使用 GSH 测定试剂盒测定谷胱甘肽（GSH）含量。
我们表明，CISD2 的敲低显着加速了 Erastin 诱导的铁死亡，伴随着过量的脂质过氧化、GSH 耗竭和铁积累，而过表达 CISD2 则阻碍了对 Erastin 的敏感性。通过共聚焦显微镜和 Western blot 进一步检测表明，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，并减轻铁死亡的表型，如加速铁积累和脂质过氧化。值得注意的是，我们发现 CISD2 耗竭细胞中 Erastin 诱导的核因子红细胞 2 相关因子 2（NRF2）的代偿性升高可以被消除。机制上，CISD2 敲低促进了自噬接头蛋白 p62 的降解，并导致 Keap1 与 NRF2 的结合亲和力增加，从而导致 NRF2 的泛素化和随后的降解增加。体外和体内强制表达 NRF2 逆转了 shCISD2 细胞对铁死亡的敏感性。相反，强制表达 Keap1 加剧了 NRF2 的降解，降低了 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。
总之，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。一种是 shCISD2 通过依赖铁蛋白自噬的铁蛋白周转增加游离铁的积累；另一种是 CISD2 耗竭诱导 p62-Keap1-NRF2 通路的抑制，导致氧化应激和铁死亡。
