Mitochondrial stress protein recognition of inactivated dehydrogenases during mammalian cell death

Abstract

The mammalian renal toxicant tetraf luoroethylcysteine (TFEC) is metabolized to a reactive intermediate that covalently modifies the lysine residues of a select group of mitochondrial proteins, forming dif luorothioamidyl lysine protein adducts. Cellular damage is initiated by this process and cell death ensues. NH 2 -terminal sequence analysis of purified mitochondrial proteins containing dif luorothioamidyl lysine adducts identified the lipoamide succinyltransferase and dihydrolipoamide dehydrogenase subunits of the ␣-ketoglutarate dehydrogenase complex (␣KGDH), a key regulatory component of oxidative metabolism, as targets for TFEC action. Adduct formation resulted in marked inhibition of ␣KGDH enzymatic activity, whereas the related pyruvate dehydrogenase complex was unmodified by TFEC and its activity was not inhibited in vivo. Covalent modification of ␣KGDH subunits also resulted in interactions with mitochondrial chaperonin HSP60 in vivo and with HSP60 and mitochondrial HSP70 in vitro. These observations confirm the role of mammalian stress proteins in the recognition of abnormal proteins and provide supporting evidence for reactive metabolite-induced cell death by modification of critical protein targets.

Mitochondrial aspartate aminotransferase catalyses cysteine S-conjugate β-lyase reactions

“…Thus disruption of the malate\aspartate shuttle in kidney might lead to serious imbalance in ion regulation. In addition, as noted in the Introduction, the E2 and E3 subunits of rat kidney OGDHC are targeted by TFEC in i o [8], and OGDHC is strongly inactivated in both PC12 and TAMH cells exposed to TFEC [9,10]. The thioacylating fragment released from TFEC is extremely reactive and is unlikely to diffuse far from its site of formation [3,5].…”

“…However, because urea is excreted by the kidneys and glucose is reabsorbed by the kidneys, cysteine S-conjugateinduced damage to the kidney is associated with increased plasma urea\blood urea nitrogen and increased glucose excretion in the urine (e.g. [8,17,18]). …”

“…Labelled mitochondrial proteins include HSP60, HSP70, mitochondrial aspartate aminotransferase (mitAspAT), lipoamide succinyltransferase [also known as E2o (equivalent to E2k, where ' o ' and ' k ' refer to ' oxo ' or ' keto ' respectively)], dihydrolipoamide dehydrogenase (E3) and aconitase [7][8][9]. TFEC administration to rats results in a time-dependent loss of kidney 2-oxoglutarate dehydrogenase complex (OGDHC) activity [8] and aconitase activity [9]. Exposure of cultured PC12 cells to TFEC results in pronounced loss of OGDHC and mitAspAT, but not of cytosolic aspartate aminotransferase (cytAspAT), activities, and alterations to the pyruvate dehydrogenase regulatory machinery [10].…”

Quantitative α-Ketoglutarate Dehydrogenase Activity Staining in Brain Sections and in Cultured Cells

“…N2a neuroblastoma cells were treated with the irreversible inhibitor, S-(1,1,2,2-tetrafluoroethy)-L-cysteine (TFEC) (18), and a reversible inhibitor, DL-␣-keto-␤-methyl-n-valeric acid (KMV). TFEC is a halogenated cysteine S-conjugate which is converted to a reactive fragment by cysteine S-conjugate ␤-lyases (19,20). The reactive fragment is a potent inhibitor of KG-DHC (18).…”

Cisplatin-Induced Toxicity Is Associated with Platinum Deposition in Mouse Kidney Mitochondria in Vivo and with Selective Inactivation of the α-Ketoglutarate Dehydrogenase Complex in LLC-PK1 Cells

“…mitAspAT is thought to be part of a metabolon that also includes KGDHC and aconitase (24). KGDHC activity is decreased in the kidneys of TFEC-treated rats (58,59) and in TFEC-treated PC12 cells (36). It was proposed that the susceptibility of KGDHC to inactivation by TFEC and to its thioacylation in rats treated with TFEC is due to close juxtapositioning of mitAspAT to KGDHC and to toxicant targeting of a reactive sulfurcontaining fragment from mitAspAT to KGDHC (24,25,59).…”