Systematic and Quantitative Investigation of Newly Synthesized Proteins Reveals Distinct Ion Homeostasis and Mitochondrial Changes between Cuproptosis and Ferroptosis in Human Cells

Dysregulated metal ion metabolism and its connection to cell death attract great attention in cell biology and biomedicine. There are two major types of known metal ion-induced cell death so far. Well-documented ferroptosis is an iron-dependent form of cell death driven by lipid peroxidation, and the recently discovered cuproptosis is copper-dependent cell death, possibly related to mitochondrial damage and cell stress. Although some studies have suggested a possible link between cuproptosis and ferroptosis, the cellular responses and mechanistic differences between these two forms of metal ion-dependent cell death remain to be explored. Here, we systematically and quantitatively analyzed newly synthesized proteins (NSPs), which reflect rapid changes in gene expression, in cells undergoing ferroptosis and cuproptosis through integrating metabolic labeling, bioorthogonal chemistry, and multiplexed proteomics. The results revealed that both types of cell death shared some common features, such as mitochondrial disorder and gene expression suppression. Furthermore, different changes between them were also uncovered. In cuproptosis, proteins related to zinc ion homeostasis were elevated because intracellular copper and zinc ions are cooperatively and competitively involved in multiple biological processes, and excess copper ions impact zinc ion homeostasis in cells. Moreover, damaged mitochondria were found to be cleared mainly through ubiquitin-mediated mitophagy. In contrast, ferroptosis is associated with an increased level of calcium-binding proteins and a compensatory upregulation of key antioxidant defense systems while concurrently showing a notable decrease in RNA alternative splicing-related proteins. Taken together, a comprehensive and comparative analysis of NSPs in cuproptosis and ferroptosis provides us with a unique opportunity to understand the molecular mechanisms of these two important forms of metal ion-dependent cell death.