ZNT1 Involves Cuproptosis through Regulating MTF1-conduced Expression of MT1X under Copper Overload

锌离子转运蛋白1参与铜死亡途径的机制研究

Industrial activities such as smelting emissions,mineral combustion and industrial wastewater discharge might lead to copper pollution in the environment.This kind of copper pollution has harmful effects on aquatic o rganisms,plants and animals through direct or indirect exposure.However,the current understanding of the toxicity of copper is rather limited.Copper overload can perturb intracellular homeostasis and induce oxidative stress and e ven cell death.Recently,cuproptosis has been identified as a copper-dependent form of cell death induced by o xidative stress in mitochondria.We uncover here that zinc transporter 1(ZNT1)is an important regulator involved in cuproptosis.Firstly,we established the copper overload-induced cell death model with the overexpression of copper importer SLC31A1 in HeLa cells.Using this model,we conducted unbiased genome-wide CRISPR-Cas9 screens in cells treated with copper.Our results revealed a significant enrichment of ZNT1 gene in both library A and library B plasmids.Knocking out of ZNT1 in HeLa cells notably prevented cuproptosis.Subsequent knockout of metal transcription factor 1(MTF1)in ZNT1-deficient cells nearly abolished their ability to resist copper-induced cell death.However,overexpression of metallothionein 1X(MT1X)in the double-knockout cells could p artially restored the resistance to cuproptosis by loss of MTF1.Mechanistically,knockout of ZNT1 could promote MT1X expression by activating MTF1.As a consequence,the interaction between MT1X and copper was e nhanced,reducing the flow of copper into mitochondria and eliminating mitochondria damage.Taken together,this study reveals the important role of ZNT1 in cuproptosis and shows MTF1-MT1X axis mediated resistance to c uproptosis.Moreover,our study will help to understand the regulatory mechanism of cellular and systemic copper homeostasis under copper overload,and present insights into novel treatments for damages caused by both genetic copper overload diseases and environmental copper contamination.

冶炼排放、矿物燃烧和工业废水排放等工业活动都可能导致环境中的铜污染。铜污染可能通过直接或间接接触对水生生物、植物和动物产生有害影响。然而,目前对铜的毒性的认识相当有限,部分研究表明铜负荷会扰乱细胞内稳态,引起氧化应激甚至细胞死亡。最近,一种新的细胞死亡形式被发现并命名为“铜死亡”,这是一种依赖铜的线粒体氧化应激诱导的细胞死亡类型。本研究发现锌离子转运蛋白1(zinc transporter 1,ZNT1)在铜死亡过程中是一个重要的调节因子。首先,研究通过在HeLa细胞中过表达铜转运载体SLC31A1蛋白,构建了铜过载细胞模型。利用该细胞模型,进行了全基因组CRISPR-Cas9筛。研究结果显示,在A和B质粒库中,ZNT1基因都显著富集,经验证,敲除HeLa细胞中的ZNT1可显著抑制铜死亡。随后在ZNT1缺失细胞中敲除金属转录因子1(metal transcription factor 1,MTF1),细胞几乎丧失了抵抗铜死亡的能力。然而,在双敲除细胞中,过表达金属硫蛋白1X(metallothionein 1X,MT1X)部分恢复对铜死亡的抗性。从机制上讲,敲除ZNT1蛋白后铜暴露会显著激活MTF1转录活性,促进MT1X的表达,此时,MT1X与自由态铜离子的相互作用增强,减少了进入线粒体的铜,消除了线粒体损伤。综上所述,本研究揭示了ZNT1在铜死亡中的重要作用,并显示了MTF1-MT1X轴介导的铜死亡抵抗机制。此外,本研究将有助于了解铜过载时细胞和全身铜稳态的调节机制,并为遗传性铜过载疾病和环境铜污染引起的铜中毒疾病提供新的治疗方法。