title: In situ kinetic trapping reveals a fingerprint of reversible protein thiol oxidation in the mitochondrial matrix.
abstract: Reactive oxygen species (ROS) are released at the mitochondrial inner membrane by the electron transport chain (ETC). Increasing evidence suggests that mitochondrial H2O2 acts as a signaling molecule and participates in the (feedback) regulation of mitochondrial activity and turnover. It seems likely that key mitochondrial components contain redox-sensitive thiols that help to adapt protein function to changes in electron flow. However, the identity of most redox-regulated mitochondrial proteins remains to be defined. Thioredoxin 2 (Trx2) is the major protein-thiol-reducing oxidoreductase in the mitochondrial matrix. We used in situ mechanism-based kinetic trapping to identify disulfide-exchange interactions of Trx2 within functional mitochondria of intact cells. Mass spectrometry successfully identified known and suspected Trx2 target proteins and, in addition, revealed a set of new candidate target proteins. Our results suggest that the mitochondrial protein biosynthesis machinery is a major target of ETC-derived ROS. In particular, we identified mitochondrial methionyl-tRNA synthetase (mtMetRS) as one of the most prominent Trx2 target proteins. We show that an increase in ETC-derived oxidants leads to an increase in mtMetRS oxidation in intact cells. In conclusion, we find that in situ kinetic trapping provides starting points for future functional studies of intramitochondrial redox regulation.
authors.affiliation: Division of Redox Regulation, DKFZ-ZMBH Alliance, German Cancer Research Center, 69120 Heidelberg, Germany.
authors.forename: Johanna
authors.initials: J
authors.lastname: Engelhard
authors.forename: Brooke E
authors.initials: BE
authors.lastname: Christian
authors.forename: Lars
authors.initials: L
authors.lastname: Weingarten
authors.forename: Gabriele
authors.initials: G
authors.lastname: Kuntz
authors.forename: Linda L
authors.initials: LL
authors.lastname: Spremulli
authors.forename: Tobias P
authors.initials: TP
authors.lastname: Dick
mesh_terms: D002478:Cells, Cultured; D003001:Cloning, Molecular; D004579:Electron Transport; D006801:Humans; D007700:Kinetics; D008718:Methionine-tRNA Ligase / Q000302:isolation & purification / Q000378:metabolism; D008928:Mitochondria / Q000201:enzymology / Q000378:metabolism*; D024101:Mitochondrial Proteins / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism; D010084:Oxidation-Reduction; D017382:Reactive Oxygen Species / Q000378:metabolism; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism; D013438:Sulfhydryl Compounds / Q000378:metabolism*; D013879:Thioredoxins / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism
chemical_list: D024101:Mitochondrial Proteins; D017382:Reactive Oxygen Species; D011994:Recombinant Proteins; D013438:Sulfhydryl Compounds; C467002:TXN2 protein, human; D013879:Thioredoxins; D008718:Methionine-tRNA Ligase