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Mitochondrial stress protein recognition of inactivated dehydrogenases during mammalian cell death
S A Bruschi1, J G Lindsay, J W Crabb
1Department of Medicinal Chemistry, University of Washington, Seattle, WA 98195-7610, USA. sambru@u.washington.edu
Abstract:
The mammalian renal toxicant tetrafluoroethylcysteine (TFEC) is metabolized to a reactive intermediate that covalently modifies the lysine residues of a select group of mitochondrial proteins, forming difluorothioamidyl lysine protein adducts. Cellular damage is initiated by this process and cell death ensues. NH2-terminal sequence analysis of purified mitochondrial proteins containing difluorothioamidyl lysine adducts identified the lipoamide succinyltransferase and dihydrolipoamide dehydrogenase subunits of the alpha-ketoglutarate dehydrogenase complex (alphaKGDH), a key regulatory component of oxidative metabolism, as targets for TFEC action. Adduct formation resulted in marked inhibition of alphaKGDH enzymatic activity, whereas the related pyruvate dehydrogenase complex was unmodified by TFEC and its activity was not inhibited in vivo. Covalent modification of alphaKGDH 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.
Insights
The renal toxicant tetrafluoroethylcysteine (TFEC) modifies mitochondrial proteins, specifically the alpha-ketoglutarate dehydrogenase complex (alphaKGDH). This adduct formation inhibits alphaKGDH activity, leading to cellular damage and death.
Area of Science:
- Biochemistry
- Toxicology
- Cell Biology
Background:
- Mammalian renal toxicant tetrafluoroethylcysteine (TFEC) causes cellular damage.
- TFEC is metabolized into reactive intermediates that modify proteins.
Purpose of the Study:
- Identify specific mitochondrial protein targets of TFEC.
- Investigate the functional consequences of TFEC-induced protein modification.
- Elucidate the role of stress proteins in response to TFEC toxicity.
Main Methods:
- NH2-terminal sequence analysis of purified mitochondrial proteins.
- Enzymatic activity assays for alpha-ketoglutarate dehydrogenase complex (alphaKGDH) and pyruvate dehydrogenase complex.
- In vivo and in vitro interaction studies with heat shock proteins (HSP60, HSP70).
Main Results:
- TFEC forms difluorothioamidyl lysine adducts on lipoamide succinyltransferase and dihydrolipoamide dehydrogenase subunits of alphaKGDH.
- Adduct formation significantly inhibits alphaKGDH enzymatic activity.
- Pyruvate dehydrogenase complex remained unaffected.
- Modified alphaKGDH subunits interacted with HSP60 and HSP70.
Conclusions:
- TFEC specifically targets and inhibits the alpha-ketoglutarate dehydrogenase complex (alphaKGDH), a key metabolic enzyme.
- Modification of alphaKGDH leads to cellular damage and death.
- Mammalian stress proteins are involved in recognizing TFEC-modified proteins.