The redox-active CXXC motif of BCAT1 regulates extracellular vesicle biogenesis in U937 macrophages

Cytosolic branched-chain aminotransferase 1 (BCAT1) is a canonical transaminase that catalyses the breakdown of essential amino acids, including leucine, isoleucine and valine. Our previous work showed that BCAT1 also contains a redox-active CXXC motif with antioxidant activity, reducing intracellular reactive oxygen species (ROS) in the monocytic U937 cell line. Moreover, these cells display decreased expression of CD14/CD68 compared to controls, a phenotype indicative of M2 macrophage polarisation. Given that Extracellular Vesicle (EV) release is associated with oxidative stress, and that there is increasing interest in the therapeutic use of M1-and M2-derived macrophage EVs, we investigated whether the BCAT1 CXXC motif could modulate EV release in U937 cells constitutively overexpressing WT BCAT1, a CXXC motif mutant (CXXS BCAT1), or vector control. Here, we show that whilst WT BCAT1 cells display significantly lower intracellular ROS compared to controls, nanoparticle tracking analysis demonstrates that WT BCAT1 significantly increases EV generation compared to CXXS BCAT1 and vector controls under both normal (10% FBS) and serum-starved (0% FBS) conditions. Analysis of CD63/CD81/CD9 tetraspanin composition revealed that the majority of EVs produced were CD63 + , with serum starvation further increasing CD63 + EV abundance. Taken together, these data indicate that BCAT1, and its redox-active CXXC motif, may play a novel role in EV biogenesis in macrophage-like cells.