Fig 1: Mechanistic insights into the biological activity of SSC in CHO cells. (A) Chemical interaction between vitamin B12 and sulfur containing compounds. Upon incubation of cyanocobalamin with increasing amounts of either SSC, sulfate, sulfite, thiosulfate or tetrathionate, only sulfite was shown to interact with the vitamin leading to the formation of sulfitocobalamin. (B) Effect of sulfur containing compounds on the CHO intracellular redox potential and protective effect towards a peroxide insult. Sulfite was effective at decreasing the cellular redox potential and at protecting the cells upon hydrogen peroxide insult. (C) Relative quantification of TST and ETHE1 using capillary-based Western Blot showing a higher sulfur scavenging capacity upon SSC treatment. (D) Capacity of oxidoreductases GR, GRX1, TRX1, TRXR to reduce GS-SO3 in presence of catalytical amounts of GSH and NADPH reducing equivalent. Only the combination GRX1/GR/GSH/NADPH was able to reduce GS-SO3 leading to the formation of GSH and oxidized NADP+. (E) Effect of GS-SO3 on GGT activity. GGT activity was measured using a commercial activity kit applying γ-Glu-pNa as a substrate. Untargeted LC-MS enabled the relative quantification of the product of the reaction, SSC-Gly. (F) Schematical representation of the fate of GS-SO3 either intracellularly (mediated by oxidoreductases), or extracellularly (mediated by GGT). Results are presented as mean ± SEM of 3 independent experiments.
Fig 2: A detailed view of the SSC metabolization in CHO cells using a multi-omics approach. Small molecules and proteins (outlined in black) passing the pre-defined criteria (AUC fold change: ± 1.5 or ± 1.1, OPLS-DA significance test) are represented by colors, and highlighted in red (upregulation) or green (downregulation). (1) After SSC uptake via the CysS/Glu antiporter (xc −), SSC chemically interacts with GSH, leading to the glutathione mixed disulfides GS-SO3 and GS-Cys. GRX1-mediated reduction of GS-SO3 increases the sulfur species pool, among which sulfite further reacts with vitamin B12 to sulfitocobalamin and decreases the cellular redox potential. GS-SO3 is hypothesized to be further exported via the MRP1 transporter, upon which the γ-glutamyl moiety of GS-SO3 is cleaved by GGT. Formed SSC-Gly peptides are suggested to be cleaved by a dipeptidase, releasing SSC for re-utilization. At the same time, the GS-Cys reduction reaction results in an increased intracellular Cys availability, which in turn leads to a decreased xc − expression to maintain cellular homeostasis. (2) Cys cellular resources are directed towards GSH synthesis, at the expense of γ-glutamyl-peptide synthesis, which might require more glutamate consumption whose synthesis might be promoted by the transamination of alanine by ALT. (3) Other Cys catabolic pathways include the taurine and presumably the Fe-S cluster synthesis pathway, the latter of which correlates with an increased expression of Fe-S-containing proteins involved in the TCA cycle and oxidative phosphorylation as well as the absence of the iron starvation response (indicated by a decrease in TFRC). Increasing ETHE1 and TST protein levels, involved in the sulfur species catabolism via the mitochondrial sulfide oxidation pathway, might be required to scavenge the formed intracellular sulfur containing species, mainly sulfite and thiosulfate. AA: amino acid, Ala: alanine, ALT: alanine aminotransferase, CAT: cysteine aminotransferase, CDO: cysteine dioxygenase, CoQ: coenzyme Q, CSAD: cysteine sulfinic acid decarboxylase, Cys: cysteine, ETHE1: ethylmalonic encephalopathy protein 1, Fe-S cluster: iron-sulfur cluster, FMO1: flavin-containing monooxygenase 1, GCLC: glutamate cysteine ligase (catalytic subunit), GGT: γ-glutamyltransferase, Glu: glutamate, Gly: glycine, GR: glutathione reductase, GRX1: glutaredoxin 1, GS-Cys: cysteine glutathione, GSH: glutathione (reduced), GSS: glutathione synthetase, GSSG: glutathione (oxidized), GSSH: glutathione persulfide, GS-SO3: sulfo-glutathione, H2S: hydrogen sulfide, MPST: 3-mercaptopyruvate sulfurtransferase, MRP1: multidrug resistance protein 1, Pyr: pyruvate, S2O3 2−: thiosulfate, S4O6 2−: tetrathionate, SO3 2−: sulfite, SO4 2−: sulfate, SQR: sulfide:quinone oxidoreductase, SSC: S-sulfocysteine, SUOX: sulfite oxidase, TFRC1: transferrin receptor protein 1, TST: thiosulfate sulfurtransferase, xc −: CysS/Glu antiporter, α-KG: α-ketoglutarate, γ-Glu: γ-glutamyl moiety.
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