title: Mitochondrial aconitase modification, functional inhibition, and evidence for a supramolecular complex of the TCA cycle by the renal toxicant S-(1,1,2,2-tetrafluoroethyl)-L-cysteine.
abstract: Metabolism of the common industrial gas tetrafluoroethylene in mammals results in the formation of S-(1,1,2,2)-tetrafluoroethyl-L-cysteine (TFEC), which can be bioactivated by a mitochondrial C-S lyase commonly referred to as beta-lyase. The resultant "reactive intermediate", difluorothioacetyl fluoride (DFTAF), is a potent thioalkylating and protein-modifying species. Previously, we have identified mitochondrial HSP70, HSP60, aspartate aminotransferase, and the E2 and E3 subunits of the alpha-ketoglutarate dehydrogenase (alphaKGDH) complex as specific proteins structurally modified during this process. Moreover, functional alterations to the alphaKGDH complex were also detected and implicated in the progression of injury. We report here the identification, by tandem mass spectrometry, and functional characterization of the final remaining major protein species modified by DFTAF, previously designated as P99(unk), as mitochondrial aconitase. Aconitase activity was maximally inhibited by 56.5% in renal homogenates after a 6 h exposure to TFEC. In comparison to alphaKGDH, aconitase inhibition (up to 79%) in a cell culture model for TFEC-mediated cytotoxicity was greater and preceded alphaKGDH inhibition, indicating that aconitase modification may constitute an early event in TFEC-mediated mitochondrial damage and cell death. These findings largely define the initial lesion of TFEC-mediated cell death and also have implications for the modeling of mitochondrial enzymatic architecture and the localization and identity of renal mitochondrial cysteine S-conjugate beta-lyase.
authors.affiliation: Department of Medicinal Chemistry, University of Washington, Seattle, WA 98195, USA.
authors.forename: Eric A
authors.initials: EA
authors.lastname: James
authors.forename: Steven P
authors.initials: SP
authors.lastname: Gygi
authors.forename: Michael L
authors.initials: ML
authors.lastname: Adams
authors.forename: Robert H
authors.initials: RH
authors.lastname: Pierce
authors.forename: Nelson
authors.initials: N
authors.lastname: Fausto
authors.forename: Ruedi H
authors.initials: RH
authors.lastname: Aebersold
authors.forename: Sidney D
authors.initials: SD
authors.lastname: Nelson
authors.forename: Sam A
authors.initials: SA
authors.lastname: Bruschi
mesh_terms: D000154:Aconitate Hydratase / Q000037:antagonists & inhibitors*; D000818:Animals; D013437:Carbon-Sulfur Lyases / Q000378:metabolism; D002952:Citric Acid Cycle / Q000187:drug effects*; D003545:Cysteine / Q000031:analogs & derivatives* / Q000378:metabolism / Q000494:pharmacology*; D006845:Hydrocarbons, Fluorinated / Q000378:metabolism / Q000494:pharmacology*; D007655:Ketoglutarate Dehydrogenase Complex / Q000037:antagonists & inhibitors*; D007668:Kidney / Q000187:drug effects*; D008297:Male; D051379:Mice; D008928:Mitochondria / Q000187:drug effects* / Q000201:enzymology; D051381:Rats; D011916:Rats, Inbred F344; D014407:Tumor Cells, Cultured; D005466:Fluorocarbons
chemical_list: D000154:Aconitate Hydratase; D013437:Carbon-Sulfur Lyases; D003545:Cysteine; D006845:Hydrocarbons, Fluorinated; D007655:Ketoglutarate Dehydrogenase Complex; C064116:S-(1,1,2,2-tetrafluoroethyl)cysteine; D005466:Fluorocarbons; C108902:cysteine-S-conjugate beta-lyase