Abstract

Cuproptosis, a copper-induced form of regulated cell death, holds therapeutic promise in cancer but remains mechanistically unclear. We developed Mito-TPCA, a mitochondrial thermal proximity coaggregation strategy combining enzyme-catalyzed proteome labeling with thermal profiling, to map mitochondrial protein-protein interaction dynamics during cuproptosis. This approach revealed that copper disrupts the association of pyruvate dehydrogenase kinases (PDKs) with the pyruvate dehydrogenase (PDH) complex by targeting lipoyl domains, triggering PDH dephosphorylation and aberrant activation. We demonstrate that this PDH activation is a key driver of cuproptosis and contributes to the heightened susceptibility of cancer cells. These findings establish PDH dephosphorylation/activation as a central mechanism of cuproptosis and a potential anti-cancer therapeutic target. Mito-TPCA offers a versatile platform to study mitochondrial protein complex dynamics in live cells.