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锌离子转运蛋白1参与铜死亡途径的机制研究

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吴越, 杨颋芸, 闫博, 艾有为, 陈芳, 马娟, 刘思金. 锌离子转运蛋白1参与铜死亡途径的机制研究[J]. 生态毒理学报, 2024, 19(4): 53-70. doi: 10.7524/AJE.1673-5897.20240309001
引用本文: 吴越, 杨颋芸, 闫博, 艾有为, 陈芳, 马娟, 刘思金. 锌离子转运蛋白1参与铜死亡途径的机制研究[J]. 生态毒理学报, 2024, 19(4): 53-70. doi: 10.7524/AJE.1673-5897.20240309001
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Wu Yue, Yang Tingyun, Yan Bo, Ai Youwei, Chen Fang, Ma Juan, Liu Sijin. ZNT1 Involves Cuproptosis through Regulating MTF1-conduced Expression of MT1X under Copper Overload[J]. Asian Journal of Ecotoxicology, 2024, 19(4): 53-70. doi: 10.7524/AJE.1673-5897.20240309001
Citation: Wu Yue, Yang Tingyun, Yan Bo, Ai Youwei, Chen Fang, Ma Juan, Liu Sijin. ZNT1 Involves Cuproptosis through Regulating MTF1-conduced Expression of MT1X under Copper Overload[J]. Asian Journal of Ecotoxicology, 2024, 19(4): 53-70. doi: 10.7524/AJE.1673-5897.20240309001
shu

锌离子转运蛋白1参与铜死亡途径的机制研究

  • 1. 中国科学院生态环境研究中心, 环境化学与生态毒理学国家重点实验室, 北京 100085;

  • 2. 中国科学院大学, 北京 100049;

  • 3. 中国科学院遗传与发育生物学研究所, 北京 100101;

  • 4. 山东第一医科大学, 山东省医学科学院科技创新中心, 济南 250117

    • 作者简介: 吴越(1998-)，女，硕士研究生，研究方向为环境毒理学，E-mail:yuewuhj@126.com
    通讯作者: 陈芳，E-mail:fangchen@genetics.ac.cn;  马娟，E-mail:juanm@rcees.ac.cn; 
  • 基金项目:

    国家自然科学基金资助项目(22076210，22150006，22021003)；中国科学院青年创新促进会资助项目(2022042)；国家重点研发计划(2018YFA0901101，2021YFE0101500)

  • 中图分类号: X171.5

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

  • 1. State Key Laboratory of Environmental Chemistry and Ecotoxicology, Research Center for Eco-Environmental Sciences, Chinese Academy of Sciences, Beijing 100085, China;

  • 2. University of Chinese Academy of Sciences, Beijing 100049, China;

  • 3. Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, Beijing 100101, China;

  • 4. Science and Technology Innovation Center, Shandong First Medical University & Shandong Academy of Medical Sciences, Jinan 250117, China

    • Corresponding authors: Chen Fang ;  Ma Juan ; 
  • Fund Project: This work was supported under grants from the National Natural Science Foundation of China (grant numbers: 22076210, 22150006, and 22021003), the Youth Innovation Promotion Association of Chinese Academy of Sciences (2022042), the National Key Research and Development Program of China (grant number: 2018YFA0901101 and 2021YFE0101500).

  • 摘要: 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。
    Abstract: 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 organisms, 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 even cell death. Recently, cuproptosis has been identified as a copper-dependent form of cell death induced by oxidative 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 partially 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 enhanced, 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 cuproptosis. 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.
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  • 收稿日期:  2024-03-09
吴越, 杨颋芸, 闫博, 艾有为, 陈芳, 马娟, 刘思金. 锌离子转运蛋白1参与铜死亡途径的机制研究[J]. 生态毒理学报, 2024, 19(4): 53-70. doi: 10.7524/AJE.1673-5897.20240309001
引用本文: 吴越, 杨颋芸, 闫博, 艾有为, 陈芳, 马娟, 刘思金. 锌离子转运蛋白1参与铜死亡途径的机制研究[J]. 生态毒理学报, 2024, 19(4): 53-70. doi: 10.7524/AJE.1673-5897.20240309001
shu
Wu Yue, Yang Tingyun, Yan Bo, Ai Youwei, Chen Fang, Ma Juan, Liu Sijin. ZNT1 Involves Cuproptosis through Regulating MTF1-conduced Expression of MT1X under Copper Overload[J]. Asian Journal of Ecotoxicology, 2024, 19(4): 53-70. doi: 10.7524/AJE.1673-5897.20240309001
Citation: Wu Yue, Yang Tingyun, Yan Bo, Ai Youwei, Chen Fang, Ma Juan, Liu Sijin. ZNT1 Involves Cuproptosis through Regulating MTF1-conduced Expression of MT1X under Copper Overload[J]. Asian Journal of Ecotoxicology, 2024, 19(4): 53-70. doi: 10.7524/AJE.1673-5897.20240309001
shu

锌离子转运蛋白1参与铜死亡途径的机制研究

    通讯作者: 陈芳，E-mail:fangchen@genetics.ac.cn;  马娟，E-mail:juanm@rcees.ac.cn; 
    作者简介: 吴越(1998-)，女，硕士研究生，研究方向为环境毒理学，E-mail:yuewuhj@126.com
  • 1. 中国科学院生态环境研究中心, 环境化学与生态毒理学国家重点实验室, 北京 100085;
  • 2. 中国科学院大学, 北京 100049;
  • 3. 中国科学院遗传与发育生物学研究所, 北京 100101;
  • 4. 山东第一医科大学, 山东省医学科学院科技创新中心, 济南 250117
  • 收稿日期:  2024-03-09
基金项目:

国家自然科学基金资助项目(22076210，22150006，22021003)；中国科学院青年创新促进会资助项目(2022042)；国家重点研发计划(2018YFA0901101，2021YFE0101500)

摘要: 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。

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