Lawrence L. Hirschberger

Cysteine dioxygenase: a robust system for regulation of cellular cysteine levels.

Cysteine catabolism in mammals is dependent upon cysteine dioxygenase (CDO), an enzyme that adds molecular oxygen to the sulfur of cysteine, converting the thiol to a sulfinic acid known as cysteinesulfinic acid (3-sulfinoalanine). CDO is one of the most highly regulated metabolic enzymes responding to diet that is known. It undergoes up to 45-fold changes in concentration and up to 10-fold changes in catalytic efficiency. This provides a remarkable responsiveness of the cell to changes in sulfur amino acid availability: the ability to decrease CDO activity and conserve cysteine when cysteine is scarce and to rapidly increase CDO activity and catabolize cysteine to prevent cytotoxicity when cysteine supply is abundant. CDO in both liver and adipose tissues responds to changes in dietary intakes of protein and/or sulfur amino acids over a range that encompasses the requirement level, suggesting that cysteine homeostasis is very important to the living organism.

Discovery and Characterization of a Second Mammalian Thiol Dioxygenase, Cysteamine Dioxygenase

There are only two known thiol dioxygenase activities in mammals, and they are ascribed to the enzymes cysteine dioxygenase (CDO) and cysteamine (2-aminoethanethiol) dioxygenase (ADO). Although many studies have been dedicated to CDO, resulting in the identification of its gene and even characterization of the tertiary structure of the protein, relatively little is known about cysteamine dioxygenase. The failure to identify the gene for this protein has significantly hampered our understanding of the metabolism of cysteamine, a product of the constitutive degradation of coenzyme A, and the synthesis of taurine, the final product of cysteamine oxidation and the second most abundant amino acid in mammalian tissues. In this study we identified a hypothetical murine protein homolog of CDO (hereafter called ADO) that is encoded by the gene Gm237 and belongs to the DUF1637 protein family. When expressed as a recombinant protein, ADO exhibited significant cysteamine dioxygenase activity in vitro. The reaction was highly specific for cysteamine; cysteine was not oxidized by the enzyme, and structurally related compounds were not competitive inhibitors of the reaction. When overexpressed in HepG2/C3A cells, ADO increased the production of hypotaurine from cysteamine. Similarly, when endogenous expression of the human ADO ortholog C10orf22 in HepG2/C3A cells was reduced by RNA-mediated interference, hypotaurine production decreased. Western blots of murine tissues with an antibody developed against ADO showed that the protein is ubiquitously expressed with the highest levels in brain, heart, and skeletal muscle. Overall, these data suggest that ADO is responsible for endogenous cysteamine dioxygenase activity.

Mechanisms involved in the regulation of key enzymes of cysteine metabolism in rat liver in vivo

Little is known about mechanisms of regulation of cysteine dioxygenase (CDO), gamma-glutamylcysteine synthetase (GCS), and cysteine-sulfinate decarboxylase (CSDC) in response to diet. Enzyme activity and Western and Northern or dot blot analyses were conducted on liver samples from rats fed a basal low-protein diet or diets with graded levels of protein or methionine for 2 wk. Higher levels of CDO activity and CDO protein but not of CDO mRNA were observed in liver of rats fed methionine or protein-supplemented diets, indicating that CDO activity is regulated by changes in enzyme concentration. Lower concentrations of the heavy or catalytic subunit of GCS (GCS-HS) mRNA and protein, as well as a lower activity state of GCS-HS in rats fed methionine- or protein-supplemented diets, indicated that dietary regulation of GCS occurs by both pretranslational and posttranslational mechanisms. Lower CSDC activity, CSDC protein concentration, and CSDC mRNA concentration were found in rats fed the highest level of protein, and regulation appeared to involve changes in mRNA concentration. Regulation of key enzymes of cysteine metabolism in response to diet determines the use of cysteine for synthesis of its essential metabolites.Little is known about mechanisms of regulation of cysteine dioxygenase (CDO), γ-glutamylcysteine synthetase (GCS), and cysteine-sulfinate decarboxylase (CSDC) in response to diet. Enzyme activity and Western and Northern or dot blot analyses were conducted on liver samples from rats fed a basal low-protein diet or diets with graded levels of protein or methionine for 2 wk. Higher levels of CDO activity and CDO protein but not of CDO mRNA were observed in liver of rats fed methionine or protein-supplemented diets, indicating that CDO activity is regulated by changes in enzyme concentration. Lower concentrations of the heavy or catalytic subunit of GCS (GCS-HS) mRNA and protein, as well as a lower activity state of GCS-HS in rats fed methionine- or protein-supplemented diets, indicated that dietary regulation of GCS occurs by both pretranslational and posttranslational mechanisms. Lower CSDC activity, CSDC protein concentration, and CSDC mRNA concentration were found in rats fed the highest level of protein, and regulation appeared to involve changes in mRNA concentration. Regulation of key enzymes of cysteine metabolism in response to diet determines the use of cysteine for synthesis of its essential metabolites.

Synthesis of Amino Acid Cofactor in Cysteine Dioxygenase Is Regulated by Substrate and Represents a Novel Post-translational Regulation of Activity

Cysteine dioxygenase (CDO) catalyzes the conversion of cysteine to cysteinesulfinic acid and is important in the regulation of intracellular cysteine levels in mammals and in the provision of oxidized cysteine metabolites such as sulfate and taurine. Several crystal structure studies of mammalian CDO have shown that there is a cross-linked cofactor present in the active site of the enzyme. The cofactor consists of a thioether bond between the γ-sulfur of residue cysteine 93 and the aromatic side chain of residue tyrosine 157. The exact requirements for cofactor synthesis and the contribution of the cofactor to the catalytic activity of the enzyme have yet to be fully described. In this study, therefore, we explored the factors necessary for cofactor biogenesis in vitro and in vivo and examined what effect cofactor formation had on activity in vitro. Like other cross-linked cofactor-containing enzymes, formation of the Cys-Tyr cofactor in CDO required a transition metal cofactor (Fe2+) and O2. Unlike other enzymes, however, biogenesis was also strictly dependent upon the presence of substrate. Cofactor formation was also appreciably slower than the rates reported for other enzymes and, indeed, took hundreds of catalytic turnover cycles to occur. In the absence of the Cys-Tyr cofactor, CDO possessed appreciable catalytic activity, suggesting that the cofactor was not essential for catalysis. Nevertheless, at physiologically relevant cysteine concentrations, cofactor formation increased CDO catalytic efficiency by ∼10-fold. Overall, the regulation of Cys-Tyr cofactor formation in CDO by ambient cysteine levels represents an unusual form of substrate-mediated feed-forward activation of enzyme activity with important physiological consequences.

HepG2/C3A cells respond to cysteine deprivation by induction of the amino acid deprivation/integrated stress response pathway

To further define genes that are differentially expressed during cysteine deprivation and to evaluate the roles of amino acid deprivation vs. oxidative stress in the response to cysteine deprivation, we assessed gene expression in human hepatoma cells cultured in complete or cysteine-deficient medium. Overall, C3A cells responded to cysteine deprivation by activation of the eukaryotic initiation factor (eIF)2alpha kinase-mediated integrated stress response to inhibit global protein synthesis; increased expression of genes containing amino acid response elements (ASNS, ATF3, CEBPB, SLC7A11, and TRIB3); increased expression of genes for amino acid transporters (SLC7A11, SLC1A4, and SLC3A2), aminoacyl-tRNA synthetases (CARS), and, to a limited extent, amino acid metabolism (ASNS and CTH); increased expression of genes that act to suppress growth (STC2, FOXO3A, GADD45A, LNK, and INHBE); and increased expression of several enzymes that favor glutathione synthesis and maintenance of protein thiol groups (GCLC, GCLM, SLC7A11, and TXNRD1). Although GCLC, GCLM, SLC7A11, HMOX, and TXNRD1 were upregulated, most genes known to be upregulated via oxidative stress were not affected by cysteine deprivation. Because most genes known to be upregulated in response to eIF2alpha phosphorylation and activating transcription factor 4 (ATF4) synthesis were differentially expressed in response to cysteine deprivation, it is likely that many responses to cysteine deprivation are mediated, at least in part, by the general control nondepressible 2 (GCN2)/ATF4-dependent integrated stress response. This conclusion was supported by the observation of similar differential expression of a subset of genes in response to leucine deprivation. A consequence of sulfur amino acid restriction appears to be the upregulation of the cellular capacity to cope with oxidative and chemical stresses via the integrated stress response.

Knockout of the murine cysteine dioxygenase gene results in severe impairment in ability to synthesize taurine and an increased catabolism of cysteine to hydrogen sulfide

Cysteine homeostasis is dependent on the regulation of cysteine dioxygenase (CDO) in response to changes in sulfur amino acid intake. CDO oxidizes cysteine to cysteinesulfinate, which is further metabolized to either taurine or to pyruvate plus sulfate. To gain insight into the physiological function of CDO and the consequence of a loss of CDO activity, mice carrying a null CDO allele (CDO(+/-) mice) were crossed to generate CDO(-/-), CDO(+/-), and CDO(+/+) mice. CDO(-/-) mice exhibited postnatal mortality, growth deficit, and connective tissue pathology. CDO(-/-) mice had extremely low taurine levels and somewhat elevated cysteine levels, consistent with the lack of flux through CDO-dependent catabolic pathways. However, plasma sulfate levels were slightly higher in CDO(-/-) mice than in CDO(+/-) or CDO(+/+) mice, and tissue levels of acid-labile sulfide were elevated, indicating an increase in cysteine catabolism by cysteine desulfhydration pathways. Null mice had lower hepatic cytochrome c oxidase levels, suggesting impaired electron transport capacity. Supplementation of mice with taurine improved survival of male pups but otherwise had little effect on the phenotype of the CDO(-/-) mice. H(2)S has been identified as an important gaseous signaling molecule as well as a toxicant, and pathology may be due to dysregulation of H(2)S production. Control of cysteine levels by regulation of CDO may be necessary to maintain low H(2)S/sulfane sulfur levels and facilitate the use of H(2)S as a signaling molecule.

Regulation of cysteine dioxygenase degradation is mediated by intracellular cysteine levels and the ubiquitin-26 S proteasome system in the living rat

Mammalian metabolism of ingested cysteine is conducted principally within the liver. The liver tightly regulates its intracellular cysteine pool to keep levels high enough to meet the many catabolic and anabolic pathways for which cysteine is needed, but low enough to prevent toxicity. One of the enzymes the liver uses to regulate cysteine levels is CDO (cysteine dioxygenase). Catalysing the irreversible oxidation of cysteine, CDO protein is up-regulated in the liver in response to the dietary intake of cysteine. In the present study, we have evaluated the contribution of the ubiquitin-26 S proteasome pathway to the diet-induced changes in CDO half-life. In the living rat, inhibition of the proteasome with PS1 (proteasome inhibitor 1) dramatically stabilized CDO in the liver under dietary conditions that normally favour its degradation. Ubiquitinated CDO intermediates were also seen to accumulate in the liver. Metabolic analyses showed that PS1 had a significant effect on sulphoxidation flux secondary to the stabilization of CDO but no significant effect on the intracellular cysteine pool. Finally, by a combination of in vitro hepatocyte culture and in vivo whole animal studies, we were able to attribute the changes in CDO stability specifically to cysteine rather than the metabolite 2-mercaptoethylamine (cysteamine). The present study represents the first demonstration of regulated ubiquitination and degradation of a protein in a living mammal, inhibition of which had dramatic effects on cysteine catabolism.

Determination of cysteinesulfinate, hypotaurine and taurine in physiological samples by reversed-phase high-performance liquid chromatography

Cysteinesulfinate, hypotaurine and taurine, which are key metabolites of cysteine, can be separated from each other and other closely eluting amino acids in biological samples by reversed-phase high-performance liquid chromatography on a Waters Nova-Pak C18 column. Samples were derivatized with o-phthalaldehyde-2-mercaptoethanol prior to injection. The elution system consisted of 100 mM potassium phosphate buffer, pH 7.0, with 3% (v/v) tetrahydrofuran with an initial isocratic phase at 1.2% acetonitrile and a gradient from 1.2 to 12.8% acetonitrile. This method is suitable for measurement of the production of metabolites from cysteine by isolated cells and for analysis of plasma and tissue extracts. Low levels of hypotaurine in rat tissues were easily measured with this method and are reported here for the first time.

The Cysteine Dioxgenase Knockout Mouse: Altered Cysteine Metabolism in Nonhepatic Tissues Leads to Excess H2S/HS− Production and Evidence of Pancreatic and Lung Toxicity

AIMS To define the consequences of loss of cysteine dioxygenase (CDO) on cysteine metabolism at the tissue level, we determined levels of relevant metabolites and enzymes and evidence of H2S/HS(-) (gaseous hydrogen sulfide and its conjugate base) toxicity in liver, pancreas, kidney, and lung of CDO(-/-) mice that were fed either a taurine-free or taurine-supplemented diet. RESULTS CDO(-/-) mice had low tissue and serum taurine and hypotaurine levels and high tissue levels of cysteine, consistent with the loss of CDO. CDO(-/-) mice had elevated urinary excretion of thiosulfate, high tissue and serum cystathionine and lanthionine levels, and evidence of inhibition and destabilization of cytochrome c oxidase, which is consistent with excess production of H2S/HS(-). Accumulation of cystathionine and lanthionine appeared to result from cystathionine β-synthase (CBS)-mediated cysteine desulfhydration. Very high levels of hypotaurine in pancreas of wild-type mice and very high levels of cystathionine and lanthionine in pancreas of CDO(-/-) mice were observed, suggesting a unique cysteine metabolism in the pancreas. INNOVATION The CDO(-/-) mouse model provides new insights into tissue-specific cysteine metabolism, particularly the role of pancreas in metabolism of excess cysteine by CBS-catalyzed reactions, and will be a useful model for studying the effects of excess endogenous production of H2S/HS(-). CONCLUSION The CDO(-/-) mouse clearly demonstrates that H2S/HS(-) production in tissues can exceed the capacity of the animal to oxidize sulfide to sulfate and demonstrates that pancreas and lung are more susceptible to toxicity from endogenous H2S/HS(-)production than are liver and kidney.

Extrahepatic tissues compensate for loss of hepatic taurine synthesis in mice with liver-specific knockout of cysteine dioxygenase

Because hepatic cysteine dioxygenase (CDO) appears to play the major role in controlling cysteine catabolism in the intact rat, we characterized the effect of a lack of hepatic CDO on the regulation of cysteine and its metabolites at the whole body level. In mice with liver-specific deletion of CDO expression, hepatic and plasma cysteine levels increased. In addition, in mice with liver-specific deletion of CDO expression, the abundance of CDO and the proportion of CDO existing as the mature, more active isoform increased in extrahepatic tissues that express CDO (kidney, brown fat, and gonadal fat). CDO abundance was also increased in the pancreas, where most of the enzyme in both control and liver CDO-knockout mice was in the more active isoform. This upregulation of CDO concentration and active-site cofactor formation were not associated with an increase in CDO mRNA and thus presumably were due to a decrease in CDO degradation and an increase in CDO cofactor formation in association with increased exposure of extrahepatic tissues to cysteine in mice lacking hepatic CDO. Extrahepatic tissues of liver CDO-knockout mice also had higher levels of hypotaurine, consistent with increased metabolism of cysteine by the CDO/cysteinesulfinate decarboxylase pathway. The hepatic CDO-knockout mice were able to maintain normal levels of glutathione, taurine, and sulfate. The maintenance of taurine concentrations in liver as well as in extrahepatic tissues is particularly notable, since mice were fed a taurine-free diet and liver is normally considered the major site of taurine biosynthesis. This redundant capacity for regulation of cysteine concentrations and production of hypotaurine/taurine is additional support for the bodys robust mechanisms for control of body cysteine levels and indicates that extrahepatic tissues are able to compensate for a lack of hepatic capacity for cysteine catabolism.