Cysteine S-conjugate ß-lyases: important roles in the metabolism of naturally occurring sulfur and selenium-containing compounds, xenobiotics and anticancer agents.

Cysteine S-conjugate ß-lyases are pyridoxal 5'-phosphate-containing enzymes that catalyze ß-elimination reactions with cysteine S-conjugates that possess a good leaving group in the ß-position. The end products are aminoacrylate and a sulfur-containing fragment. The aminoacrylate tautomerizes and hydrolyzes to pyruvate and ammonia. The mammalian cysteine S-conjugate ß-lyases thus far identified are enzymes involved in amino acid metabolism that catalyze ß-lyase reactions as non-physiological side reactions. Most are aminotransferases. In some cases the lyase is inactivated by reaction products. The cysteine S-conjugate ß-lyases are of much interest to toxicologists because they play an important key role in the bioactivation (toxication) of halogenated alkenes, some of which are produced on an industrial scale and are environmental contaminants. The cysteine S-conjugate ß-lyases have been reviewed in this journal previously (Cooper and Pinto in Amino Acids 30:1-15, 2006). Here, we focus on more recent findings regarding: (1) the identification of enzymes associated with high-M(r) cysteine S-conjugate ß-lyases in the cytosolic and mitochondrial fractions of rat liver and kidney; (2) the mechanism of syncatalytic inactivation of rat liver mitochondrial aspartate aminotransferase by the nephrotoxic ß-lyase substrate S-(1,1,2,2-tetrafluoroethyl)-L-cysteine (the cysteine S-conjugate of tetrafluoroethylene); (3) toxicant channeling of reactive fragments from the active site of mitochondrial aspartate aminotransferase to susceptible proteins in the mitochondria; (4) the involvement of cysteine S-conjugate ß-lyases in the metabolism/bioactivation of drugs and natural products; and (5) the role of cysteine S-conjugate ß-lyases in the metabolism of selenocysteine Se-conjugates. This review emphasizes the fact that the cysteine S-conjugate ß-lyases are biologically more important than hitherto appreciated.

Measurement of cysteine S-conjugate ß-lyase activity.

Cysteine S-conjugate ß-lyases are pyridoxal 5'-phosphate (PLP)-containing enzymes that catalyze the conversion of cysteine S-conjugates [RSCH(2)CH(NH(3) (+))CO(2) (-)] and selenium Se-conjugates [RSeCH(2)CH(NH(3) (+))CO(2) (-)] that contain a leaving group in the ß position to pyruvate, ammonium and a sulfur-containing fragment (RSH) or selenium-containing fragment (RSeH), respectively. In mammals, at least ten PLP enzymes catalyze ß-elimination reactions with such cysteine S-conjugates. All are enzymes involved in amino acid metabolism that do not normally catalyze a ß-lyase reaction, but catalyze a non-physiological ß-lyase side-reaction that depends on the electron-withdrawing properties of the -SR or -SeR moiety. In the case of cysteine S-conjugates, if the eliminated RSH is stable, the compound may be S-thiomethylated and excreted (thiomethyl shunt) or S-glucuronidated and harmlessly excreted. However, if RSH is chemically reactive, the cysteine S-conjugate may be toxic as a result of the ß-lyase reaction. The cysteine S-conjugate ß-lyase pathway is of particular interest to toxicologists because it is involved in the bioactivation (toxification) of halogenated alkenes and certain drugs. This unit provides protocols for the analysis of cysteine S-conjugate ß-lyase activity.

Cytosolic heat shock proteins and heme oxygenase-1 are preferentially induced in response to specific and localized intramitochondrial damage by tetrafluoroethylcysteine.

Previously, S-(1,1,2,2-tetrafluoroethyl)-l-cysteine (TFEC) was shown to mediate cytotoxicity by covalently modifying a well-defined group of intramitochondrial proteins including aconitase, alpha-ketoglutarate dehydrogenase (alphaKGDH) subunits, heat shock protein 60 (HSP60) and mitochondrial HSP70 (mtHSP70). To investigate the cellular responses to this mitochondrial damage, microarray analysis of TFEC treated murine hepatocytes of the TAMH cell line was carried out. Results of these studies revealed a HSP response that was significantly stronger than other well-characterized hepatotoxicants including acetaminophen, diquat and rotenone. Specifically, cytosolic HSP25, HSP40, HSP70, HSP105 and microsomal HSP32 (HO-1) were strongly upregulated within the first few hours of TFEC treatment, while little change was observed among other HSPs that are predominantly localized in the mitochondria and endoplasmic reticulum (ER). Post-translational modification of HSP25 was also observed with the appearance of a unique DTT-resistant immunoreactive band at about 50kDa, a putative dimer. The biological significance of HSP responses to TFEC-induced toxicity were subsequently demonstrated using the "gain of function" pretreatment: heat shock. Overall, we report an atypical HSP induction profile that does not conform to changes expected of a classical temperature shock. Furthermore, despite a well-defined intramitochondrial origin of toxicity, TFEC rapidly evokes an early and strong upregulation of cytosolic stress proteins. The cytoprotective effects of such HSP responses suggest a plausible role in modulating the progression of TFEC-induced cellular injury.

Nrf2 activation involves an oxidative-stress independent pathway in tetrafluoroethylcysteine-induced cytotoxicity.

Tetrafluoroethylcysteine (TFEC), a metabolite of the industrial gas tetrafluoroethylene, can cause both nephrotoxicity and limited hepatotoxicity in animal models, and this is associated with the covalent modification of specific intramitochondrial proteins including heat shock protein 60 (HSP60), mitochondrial HSP70 (mtHSP70), aspartate aminotransferase (AST), aconitase, and alpha-ketoglutarate dehydrogenase (alphaKGDH). Using the murine TAMH cell line as a useful in vitro model for TFEC toxicity, we demonstrate a rapid and sustained induction of Nrf2, a member of the "cap-and-collar" transcription factor family, following exposure to cytotoxic concentrations of TFEC. A functional correlate was also established with the rapid translocation of cytosolic Nrf2 into the nucleus. In addition, transcriptional and translational upregulation of known Nrf2 regulated genes including glutamate cysteine ligase (GCL), both catalytic and modulatory subunits, heme oxygenase-1, and glutathione S-transferase (GST) isoforms were detected. While Nrf2 activation is often linked to perturbation of cellular thiol status and/or oxidative stress, we were unable to detect any significant depletion of cellular glutathione or oxidation of mitochondrial membrane cardiolipin or increases in reactive oxygen species (ROS). These data suggest Nrf2 activation is likely independent of classical oxidative stress or, at best, a result of a transient, low-level redox stress. Moreover, supporting evidence indicates an early endoplasmic reticular (ER) stress response after TFEC treatment, with a time-dependent upregulation of the ER responsive genes gadd34, gadd45, gadd153, and ndr1 . These findings suggest an alternative pathway for Nrf2 activation, i.e., Nrf2 phosphorylation through ER-mediated protein kinases such as PKR-like endoplasmic reticular kinase (PERK). Overall, the results implicate a role for Nrf2 in the cellular response to TFEC toxicity and suggest a previously unrecognized role for the ER in this model of mitochondrially initiated cytotoxicity.

BCL-xL overexpression effectively protects against tetrafluoroethylcysteine-induced intramitochondrial damage and cell death.

S-(1,1,2,2-Tetrafluoroethyl)-L-cysteine (TFEC), a major metabolite of the industrial gas tetrafluoroethylene, has been shown to mediate nephrotoxicity by necrosis. TFEC-induced cell death is associated with an early covalent modification of specific intramitochondrial proteins; including aconitase, alpha-ketoglutarate dehydrogenase (KGDH) subunits, HSP60 and HSP70. Previous studies have indicated that the TAMH line accurately models TFEC-induced in vivo cell death with dose- and time-dependent inhibitions of both KGDH and aconitase activities. Here, we show that the molecular pathway leading to TFEC-mediated cell death is associated with an early cytosolic to mitochondrial translocation of BAX, a pro-apoptotic member of the BCL-2 family. Immunoblot analyses indicated movement of BAX (21 kDa) to the mitochondrial fraction after exposure to a cytotoxic concentration of TFEC (250 microM). Subsequent cytochrome c release from mitochondria was also demonstrated, but only a modest increase in caspase activities was observed, suggesting a degeneration of early apoptotic signals into secondary necrosis. Significantly, TAMH cells overexpressing BCL-xL preserved cell viability even to supratoxicological concentrations of TFEC (< or =600 microM), and this cytoprotection was associated with decreased HSP70i upregulation, indicating suppression of TFEC-induced proteotoxicity. Hence, TFEC-induced necrotic cell death in the TAMH cell line is mediated by BAX and antagonized by the anti-apoptotic BCL-2 family member, BCL-xL.

Human mitochondrial and cytosolic branched-chain aminotransferases are cysteine S-conjugate beta-lyases, but turnover leads to inactivation.

The mitochondrial and cytosolic branched-chain aminotransferases (BCAT(m) and BCAT(c)) are homodimers in the fold type IV class of pyridoxal 5'-phosphate-containing enzymes that also contains D-amino acid aminotransferase and 4-amino-4-deoxychorismate lyase (a beta-lyase). Recombinant human BCAT(m) and BCAT(c) were shown to have beta-lyase activity toward three toxic cysteine S-conjugates [S-(1,1,2,2-tetrafluoroethyl)-L-cysteine, S-(1,2-dichlorovinyl)-L-cysteine, and S-(2-chloro-1,1,2-trifluoroethyl)-L-cysteine] and toward beta-chloro-L-alanine. Human BCAT(m) is a much more effective beta-chloro-L-alanine beta-lyase than two aminotransferases (cytosolic and mitochondrial isozymes of aspartate aminotransferase) previously shown to possess this activity. BCAT(m), but not BCAT(c), also exhibits measurable beta-lyase activity toward a relatively bulky cysteine S-conjugate [benzothiazolyl-L-cysteine]. Benzothiazolyl-L-cysteine, however, inhibits the L-leucine-alpha-ketoglutarate transamination reaction catalyzed by both enzymes. Inhibition was more pronounced with BCAT(m). In the presence of beta-lyase substrates and alpha-ketoisocaproate (the alpha-keto acid analogue of leucine), no transamination could be detected. Therefore, with an amino acid containing a good leaving group in the beta position, beta-elimination is greatly preferred over transamination. Both BCAT isozymes are rapidly inactivated by the beta-lyase substrates. The ratio of turnover to inactivation per monomer in the presence of toxic halogenated cysteine S-conjugates is approximately 170-280 for BCAT(m) and approximately 40-50 for BCAT(c). Mitochondrial enzymes of energy metabolism are especially vulnerable to thioacylation and inactivation by the reactive fragment released from toxic, halogenated cysteine S-conjugates such as S-(1,1,2,2-tetrafluoroethyl)-L-cysteine. The present results suggest that BCAT isozymes may contribute to the mitochondrial toxicity of these compounds by providing thioacylating fragments, but inactivation of the BCAT isozymes might also block essential metabolic pathways.

Toxic, halogenated cysteine S-conjugates and targeting of mitochondrial enzymes of energy metabolism.

Several haloalkenes are metabolized in part to nephrotoxic cysteine S-conjugates; for example, trichloroethylene and tetrafluoroethylene are converted to S-(1,2-dichlorovinyl)-L-cysteine (DCVC) and S-(1,1,2,2-tetrafluoroethyl)-L-cysteine (TFEC), respectively. Although DCVC-induced toxicity has been investigated since the 1950s, the toxicity of TFEC and other haloalkene-derived cysteine S-conjugates has been studied more recently. Some segments of the US population are exposed to haloalkenes either through drinking water or in the workplace. Therefore, it is important to define the toxicological consequences of such exposures. Most halogenated cysteine S-conjugates are metabolized by cysteine S-conjugate beta-lyases to pyruvate, ammonia, and an alpha-chloroenethiolate (with DCVC) or an alpha-difluoroalkylthiolate (with TFEC) that may eliminate halide to give a thioacyl halide, which reacts with epsilon-amino groups of lysine residues in proteins. Nine mammalian pyridoxal 5'-phosphate (PLP)-containing enzymes catalyze cysteine S-conjugate beta-lyase reactions, including mitochondrial aspartate aminotransferase (mitAspAT), and mitochondrial branched-chain amino acid aminotransferase (BCAT(m)). Most of the cysteine S-conjugate beta-lyases are syncatalytically inactivated. TFEC-induced toxicity is associated with covalent modification of several mitochondrial enzymes of energy metabolism. Interestingly, the alpha-ketoglutarate- and branched-chain alpha-keto acid dehydrogenase complexes (KGDHC and BCDHC), but not the pyruvate dehydrogenase complex (PDHC), are susceptible to inactivation. mitAspAT and BCAT(m) may form metabolons with KGDHC and BCDHC, respectively, but no PLP enzyme is known to associate with PDHC. Consequently, we hypothesize that not only do these metabolons facilitate substrate channeling, but they also facilitate toxicant channeling, thereby promoting the inactivation of proximate mitochondrial enzymes and the induction of mitochondrial dysfunction.

Mitochondrial aspartate aminotransferase catalyses cysteine S-conjugate beta-lyase reactions.

Rat liver mitochondrial aspartate aminotransferase (a homodimer) was shown to catalyse a beta-lyase reaction with three nephrotoxic halogenated cysteine S-conjugates [ S -(1,1,2,2-tetrafluoroethyl)-L-cysteine, S -(1,2-dichlorovinyl)-L-cysteine and S -(2-chloro-1,1,2-trifluoroethyl)-L-cysteine], and less effectively so with a non-toxic cysteine S-conjugate [benzothiazolyl-L-cysteine]. Transamination competes with the beta-lyase reaction, but is not favourable. The ratio of beta elimination to transamination in the presence of S -(1,1,2,2-tetrafluoroethyl)-L-cysteine and 2-oxoglutarate is >100. Syncatalytic inactivation by the halogenated cysteine S-conjugates is also observed. The enzyme turns over approx. 2700 molecules of halogenated cysteine S-conjugate on average for every monomer inactivated. Kidney mitochondria are known to be especially sensitive to toxic halogenated cysteine S-conjugates. Evidence is presented that 15-20% of the cysteine S-conjugate beta-lyase activity towards S -(1,1,2,2-tetrafluoroethyl)-L-cysteine in crude kidney mitochondrial homogenates is due to mitochondrial aspartate aminotransferase. The possible involvement of mitochondrial aspartate aminotransferase in the toxicity of halogenated cysteine S-conjugates is also discussed.

Cell culture model for acetaminophen-induced hepatocyte death in vivo.

Overdose of the popular, and relatively safe, analgesic acetaminophen (N-acetyl-p-aminophenol, APAP, paracetamol) can produce a fatal centrilobular liver injury. APAP-induced cell death was investigated in a differentiated, transforming growth factor alpha (TGFalpha)-overexpressing, hepatocyte cell line and found to occur at concentrations, and over time frames, relevant to clinical overdose situations. Coordinated multiorganellar collapse was evident during APAP-induced cytotoxicity with widespread, yet selective, protein degradation events in vitro. Cellular proteasomal activity was inhibited with APAP treatment but not with the comparatively nonhepatotoxic APAP regioisomer, N-acetyl-m-aminophenol (AMAP). Low concentrations of the proteasome-directed inhibitor MG132 (N-carbobenzoxyl-Leu-Leu-Leucinal) increased chromatin condensation and cellular stress responses preferentially in AMAP-treated cultures, suggesting a contribution of the proteasome in APAP- but not AMAP-mediated cell death. APAP-specific alterations to mitochondria were observed morphologically with evidence of mitochondrial proliferation in vitro. Biochemical alterations to cellular proteolytic events were also found in vivo, including APAP- or AMAP-mediated inhibition of caspase-3 processing. These results indicate that, although retaining some attributes of apoptosis, both APAP- and AMAP-mediated cell death have additional distinctive features consistent with longer term necrosis.

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.

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.