MT2A buffering exhaustion marks cuproptosis in severe COVID-19: Multi-omics integration, computational modeling, and experimental validation.

Abstract

BACKGROUND: Cuproptosis is a novel copper-dependent form of cell death closely associated with mitochondrial metabolism and protein lipoylation. We propose a "Copper Buffering-Execution Imbalance" hypothesis where metallothionein 2A (MT2A) exhaustion leads to free copper accumulation, activating the FDX1-PDH axis and triggering mitochondrial cuproptosis. Metallothioneins (MTs) are key regulators of copper homeostasis, but their mechanistic roles in severe infections remain unclear. METHODS: We employed a systems biology approach integrating in silico modeling and experimental validation. We analyzed multi-omics data from three independent datasets: (1) GSE158055 peripheral blood mononuclear cell (PBMC) single-cell transcriptomics (24 cuproptosis-related genes), (2) GSE145926 bronchoalveolar lavage fluid (BALF) single-cell transcriptomics (31 genes), and (3) Nie2021 lung tissue proteomics (26 proteins). Virtual knockout simulations (scTenifoldKnk) were performed including positive (MTF1) and negative (B2M) controls and seed sensitivity analysis to predict gene regulatory network perturbations. Findings were validated using an LPS-induced mouse model and bedside lung ultrasound (LUS) radiomics. RESULTS: Among 35 cuproptosis-related genes analyzed, we found: (1) MT2A showed discordant expression directions between PBMC and BALF (Wilcoxon test, P&#xa0;<&#xa0;0.05), suggesting tissue-specific regulation; (2) Single-cell pseudotime analysis reconstructed the "temporal trajectory of redox homeostasis collapse", revealing a "Biphasic Dynamic" of MT2A (Acute Upregulation -&#xa0;>&#xa0;Exhaustion); (3) In silico perturbation analysis confirmed that MT2A depletion triggers the activation of the FDX1-PDH execution axis, which was experimentally verified in the mouse model; (4) LUS radiomics entropy served as a macroscopic marker of this microscopic oxidative damage. CONCLUSIONS: This study combines computational modeling with experimental evidence to elucidate the MT2A-mediated "copper buffering-execution imbalance" network, identifying novel redox checkpoints for therapeutic intervention in severe COVID-19.