ZNT1 involves cuproptosis through regulating MTF1-conduced expression of MT1X under copper overload

Abstract

Abstract Background Copper is an essential but also toxic heavy metal. As a crucial micronutrient, copper is required for various enzymes in physiology and pathology. Meanwhile, copper overload has currently raised serious public health concerns. Copper overload can perturb intracellular homeostasis and induce oxidative stress and even cell death. More recently, cuproptosis has been identified as a copper-dependent form of cell death induced by oxidative stress in mitochondria. This mitochondrial cell death is characterized by lipoylated protein aggregation and loss of iron-sulfur cluster proteins. However, the current comprehension of the mechanisms underlying copper toxicity remains relatively limited, particularly concerning the molecular regulatory mechanism against cuproptosis. Methods We constructed HeLa-Cas9-SLC31A1 cells for Genome-wide CRISPR/Cas9 screen to identify new components in the execution of cuproptosis. Also, we established single and double knock out models to examine the influence of candidate genes– zinc transporter 1 (ZNT1) and metal-response element-binding transcription factor-1 (MTF1) on the accumulation of cellular copper. Additionally, we performed metallothionein 1X (MT1X) overexpression and zinc/copper competitive combination experiments to explore their functions in cuproptosis. This regulatory effect was further verified in a mouse model with copper-dependent liver injury. Results We uncover here that ZNT1 is an important regulator involved in cuproptosis. Mechanistically, because zinc is a direct activator of MTF1, knockout of ZNT1 enhanced intracellular zinc levels and then promoted MT1X expression by strongly driving MTF1 transcription factor. As a consequence, the interaction between MT1X and copper was strengthened, reducing the flow of copper into mitochondria and eliminating mitochondria damages. Conclusions 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 novel insights into novel treatments for damages caused by both genetic copper overload diseases and environmental copper contamination.