The BCAT1 CXXC motif modulates extracellular vesicle release in acute myeloid leukaemia cell line U937

Introduction
Extracellular Vesicle (EV) release is altered by oxidative stress which is also a feature of AML pathogenesis. The redox active protein tyrosine phosphatase 2 (SHP2) negatively regulates the biogenesis of EVs through dephosphorylation of CD63 associated syntenin. In turn, SHP2 is positively regulated by CXXC-motif containing oxidoreductase Thioredoxin. Our recent work has identified cytosolic branched-chain amino transferase (BCAT1) as a novel oxidoreductase through a redox active CXXC motif which decreases intracellular reactive oxygen species in myeloid cell line U937. Given these data we investigated the effect of the BCAT1-CXXC motif in syntenin modulated EV release in U937 cells.

Methods
Overexpression of vector control, BCAT1-WT and BCAT1-CXXS mutant protein in U937 cells was confirmed by western-blot and qPCR. Cells were cultured in EV-depleted Foetal Bovine Serum, conditioned media was collected and EVs were isolated via differential ultracentrifugation at 2000 xg for 20 min, 10,000 xg for 30 min and 120,000 xg for 70 min. EV concentration was determined by Nanoparticle Tracking Analysis (NTA). The presence of CD81, CD63, CD9 and syntenin at the single vesicle level was assessed by immunofluorescent staining using the Exoview platform.

Results
NTA revealed BCAT1-WT overexpression resulted in a 5.16-fold increase in EV release compared to the control and a 1.80-fold increase compared to BCAT1-CXXS (p< 0.0001). Size distribution analysis showed smaller average size of BCAT1-WT (145.5± 3.4nm) and BCAT1- CXXS (147.9± 15.75nm) EVs compared to control (202.7 ± 8.2nm). Immunofluorescent staining showed a 1.76-fold increase in CD63 positive EVs derived from BCAT1-WT cells compared to control and a 1.94-fold increase compared to BCAT1-CXXS. Conversely, BCAT1-WT CD63 positive EVs displayed a 0.62-fold decrease in syntenin compared to control and a 0.83-fold decrease compared to BCAT1-CXXS cells.

Summary/Conclusion
This study demonstrates BCAT1 increases EV release in U937 cells, moreover, the number of CD63 positive vesicles was increased despite a concurrent decrease in syntenin positive vesicles. Mutation of the CXXC motif increased the syntenin positive vesicle count but decreased vesicle release raising the possibility of a novel BCAT1-CXXC dependent regulatory mechanism for EV generation in AML cells.