Figure 4. CDO1 depletes cyst(e)ine, leading to its export as CSA.

(a) Schematic depicting intracellular cysteine metabolism. Following uptake of cystine via xCT, it is reduced to cysteine via the cellular antioxidant systems. Cysteine then enters glutathione synthesis mediated by GCL and GSS, or undergoes irreversible metabolism by CDO1 to CSA, and subsequently to HTAU by CSAD. (b) Time-dependent quantification (0, 1, 2, 4, 8, 24 hr) of intracellular cysteine (CYS) and cysteine sulfinic acid (CSA) concentrations following medium replenishment of CDO1^WT-expressing (red) and CDO1^Y157F-expressing (gray) A549 cells. *= CDO1^WT vs. CDO1^Y157F CYS, #= CDO1^WT vs. CDO1^Y157F CSA. N = 3 replicates/group, except 8 hr CYS for which N = 6 replicates/group. (c) Time-dependent quantification of GSH, HTAU, and TAU in the extracts from (b). N = 3 replicates/group. (d) Time-dependent quantification of the levels of (CYS)₂ and CSA in the medium from the cells from (b). N = 3 replicates/group. (e) Intracellular CSA concentration of H1581 cells following expression of control (sgCON) or CDO1-targeting sgRNAs (sgCDO1 #1 and #2) and Cas9. N = 4 replicates/group. (f) Quantification of (CYS)₂ consumption from the medium of the cells from (e). N = 4 replicates/group. (g) CSA production rate of NRF2 KO A549 cells reconstituted with either pLX317 empty (NRF2 -) or pLX317-NRF2 (NRF2 +), followed by expression of CDO1^Y157F (Y157F) or CDO1^WT (WT). N = 6 replicates/group. (h) Quantification of (CYS)₂ consumption from the medium of the cells from (g). (i) Intracellular CSA concentration in H1975 and H1299 cells expressing either pLX317 empty (-) or pLX317-NRF2^T80K (NRF2^T80K), followed by expression of CDO1^Y157F (Y157F) or CDO1^WT (WT). N = 3 replicates/group. (j) Quantification of (CYS)₂ consumption from the medium of the cells from (i). (k–m) CSA production rate of A549 (k), H1944 (l) and H322 (m) cells following expression of CDO1^Y157F (Y157F) or CDO1^WT (WT), and reconstituted with inactive KEAP1 (C273S), super repressor KEAP1 (C151S) or wild-type KEAP1 (WT). N = 3 replicates/group. For (g–m) cells were treated with 0.25 μg/ml doxycycline for 2 days prior to and during the assay and fresh medium was added 4 hr prior to sample collection.

Figure 4—figure supplement 1. CSA only accounts for a fraction of CDO1-dependent (CYS)₂ consumption.

(a) Time-dependent western blot analysis of CDO1 and β-Actin protein expression at 0, 1, 2, 4, 8, or 24 hr following media replenishment of CDO1^WT-expressing and CDO1^Y157F-expressing A549 cells. (b) Quantification of CDO1-dependent (CYS)₂ consumption and CSA production as cysteine molar equivalents. Because (CYS)₂ contains 2 molecules of cysteine, it counts as two molar equivalents. CDO1-dependent changes were determined by subtracting the rates of change in CDO1^Y157F cells from CDO1^WT cells. CSA accounts for 2–25% of CDO1-dependent (CYS)₂ consumption. N = 3 replicates/group, except NRF2 KO A549 (N = 6 replicates). Cells were treated with 0.25 µg/ml doxycycline for 2 days prior to and during the assay and fresh medium was added 4 hr prior to sample collection (b) or prior the indicated time points (a).