Jarvis, A. F. | bioRxiv Science

bioRxiv2026

Mitochondrial copper stabilizes lipoylated TCA cycle proteins to sustain metabolism and proliferation

Copper (Cu) is an essential cofactor for mitochondrial cytochrome c oxidase, yet whether it directly regulates mitochondrial metabolism beyond respiration remains unclear. Here we show that mitochondrial Cu, delivered by SLC25A3, is required to maintain the stability of lipoylated TCA cycle proteins. Loss of Slc25a3 or pharmacological Cu depletion selectively destabilized the lipoylated E2 subunits of mitochondrial dehydrogenases and the lipoylation enzymes LIPT1 and LIPT2, an effect not reproduced by acute electron transport chain inhibition. Mechanistically, we find that Cu directly engages the reduced lipoyl moiety using chemical probes and synthetic peptide approaches. Cu depletion impaired PDH and OGDH activity, rewired TCA cycle metabolism, and imposed a dependence on pyruvate carboxylase for anaplerosis. This metabolic defect depleted aspartate, suppressed mTORC1 signaling, and limited proliferation. Conversely, selective delivery of Cu to the mitochondria restored lipoylation, TCA cycle function, and cell growth. Together, these findings identify mitochondrial Cu as a structural regulator of the lipoylation machinery and reveal a direct link between Cu homeostasis and central carbon metabolism.

cell biology↗

bioRxiv2025

MAPK Pathway Inhibition Reshapes Kinase Chemical Probe Reactivity Reflecting Cellular Activation States

Despite the pivotal role of oncogenic kinases in cancer initiation, progression, and therapeutic resistance, functionally profiling their activity and conformational dynamics in live cells remains challenging. Existing methods often fail to capture inhibitor-bound structural states of kinases, particularly in clinically relevant contexts such as treatment response and acquired resistance, where genomic data alone are insufficient. Here, we use activity-based protein profiling (ABPP) to monitor composite amino acid reactivity changes - across cysteine, lysine, and carboxylic acid residues - as a functional readout of kinase conformation and activation state. Using electrophilic probes, we show that treatment of BRAFV600E-mutant melanoma cells with vemurafenib or trametinib decreases overall cysteine and lysine reactivity in BRAFV600E and MEK1/2, likely reflecting composite changes in amino acid accessibility across multiple reactive residues associated with inhibitor binding. Changing the order of probe addition and inhibitor treatment altered labeling outcomes, consistent with competitive engagement and structural stabilization. Comparative analysis of ATP-competitive BRAFV600E inhibitors vemurafenib and dabrafenib revealed distinct impacts on aspartate and glutamate labeling patterns, suggesting that ABPP can distinguish inhibitor-dependent differences in residue accessibility that may reflect distinct inhibitor-bound conformations. In inhibitor-resistant melanoma models, ABPP detected differential residue reactivity relative to parental cells, consistent with BRAF overexpression and the MEK2 Q60P activation mutation, both established mechanisms of MAPK inhibitor resistance. Moreover, global proteome analyses of cysteine and lysine reactivity upon BRAFV600E inhibition, revealed probe-accessible cysteine labeling changes in labeling on KSR2, indicating broader MAPK pathway remodeling. Together, these findings establish ABPP as a powerful chemical biology approach for investigating inhibitor-dependent changes in kinase residue accessibility, providing a framework to explore how conformational dynamics and pathway adaptation shape therapeutic response and resistance in oncogenic signaling networks.

cancer biology↗