Mitochondrial protein targets of thiol-reactive electrophiles.
Mitochondria serve a pivotal role in the regulation of apoptosis or programmed cell death. Recent studies have demonstrated that reactive electrophiles induce mitochondrion-dependent apoptosis. We hypothesize that covalent modification of specific mitochondrial proteins by reactive electrophiles serves as a trigger leading to the initiation of apoptosis. In this study, we identified protein targets of the model biotin-tagged electrophile probes N-iodoacetyl- N-biotinylhexylene-diamine (IAB) and 1-biotinamido-4-(4'-[maleimidoethylcyclohexane]carboxamido)butane (BMCC) in HEK293 cell mitochondrial fractions by liquid chromatography-tandem mass spectrometry (LC-MS-MS). These electrophiles reproducibly adducted a total of 1693 cysteine residues that mapped to 809 proteins. Protein modifications were selective in that only 438 cysteine sites in 1255 cysteinyl peptide adducts (35%) and 362 of the 809 identified protein targets (45%) were adducted by both electrophiles. Of these, approximately one-third were annotated to the mitochondria following protein database analysis. IAB initiated apoptotic events including cytochrome c release, caspase-3 activation, and poly(ADP-ribose)polymerase (PARP) cleavage, whereas BMCC did not. Of the identified targets of IAB and BMCC, 44 were apoptosis-related proteins, and adduction site specificity on these targets differed between the two probes. Differences in sites of modification between these two electrophiles may reveal alkylation sites whose modification triggers apoptosis.
Wong
Hansen L
HL
Department of Biochemistry, Vanderbilt University School of Medicine, U1213C Medical Research Building III, 465 21st Avenue South, Nashville, Tennessee 37232, USA.
Liebler
Daniel C
DC
D017209:Apoptosis; D001710:Biotin / Q000031:analogs & derivatives* / Q000633:toxicity; D002460:Cell Line; D006801:Humans; D024101:Mitochondrial Proteins / Q000378:metabolism*; D013438:Sulfhydryl Compounds / Q000378:metabolism
C479840:1-biotinamido-4-(4'-(maleimidomethyl)cyclohexanecarboxamido)butane; D024101:Mitochondrial Proteins; D013438:Sulfhydryl Compounds; D001710:Biotin; C043329:N-iodoacetyl-N'-biotinylhexylenediamine