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To gain further insights into the effects of elevated cysteine levels on energy metabolism and the possible mechanisms underlying these effects, we conducted studies in cysteine dioxygenase (Cdo1)-null mice. Cysteine dioxygenase (CDO) catalyzes the first step of the major pathway for cysteine catabolism. When CDO is absent, tissue and plasma cysteine levels are elevated, resulting in enhanced flux of cysteine through desulfhydration reactions. When Cdo1-null mice were fed a high-fat diet, they gained more weight than their wild-type controls, regardless of whether the diet was supplemented with taurine. Cdo1-null mice had markedly lower leptin levels, higher feed intakes, and markedly higher abundance of hepatic stearoyl-CoA desaturase 1 (SCD1) compared to wild-type control mice, and these differences were not affected by the fat or taurine content of the diet. Thus, reported associations of elevated cysteine levels with greater weight gain and with elevated hepatic Scd1 expression are also seen in the Cdo1-null mouse model. Hepatic accumulation of acylcarnitines suggests impaired mitochondrial β-oxidation of fatty acids in Cdo1-null mice. The strong associations of elevated cysteine levels with excess H2 S production and impairments in energy metabolism suggest that H2 S signaling could be involved., (© 2016 New York Academy of Sciences.)

The oxidation of cysteine in mammalian cells occurs by two routes: a highly regulated direct oxidation pathway in which the first step is catalyzed by cysteine dioxygenase (CDO) and by desulfhydration-oxidation pathways in which the sulfur is released in a reduced oxidation state. To assess the effect of a lack of CDO on production of hydrogen sulfide (H2S) and thiosulfate (an intermediate in the oxidation of H2S to sulfate) and to explore the roles of both cystathionine γ-lyase (CTH) and cystathionine β-synthase (CBS) in cysteine desulfhydration by liver, we investigated the metabolism of cysteine in hepatocytes isolated from Cdo1-null and wild-type mice. Hepatocytes from Cdo1-null mice produced more H2S and thiosulfate than did hepatocytes from wild-type mice. The greater flux of cysteine through the cysteine desulfhydration reactions catalyzed by CTH and CBS in hepatocytes from Cdo1-null mice appeared to be the consequence of their higher cysteine levels, which were due to the lack of CDO and hence lack of catabolism of cysteine by the cysteinesulfinate-dependent pathways. Both CBS and CTH appeared to contribute substantially to cysteine desulfhydration, with estimates of 56 % by CBS and 44 % by CTH in hepatocytes from wild-type mice, and 63 % by CBS and 37 % by CTH in hepatocytes from Cdo1-null mice.

Using HepG2/C3A cells and MEFs, we investigated whether induction of GSH synthesis in response to sulfur amino acid deficiency is mediated by the decrease in cysteine levels or whether it requires a decrease in GSH levels per se. Both the glutamate-cysteine ligase catalytic (GCLC) and modifier (GCLM) subunit mRNA levels were upregulated in response to a lack of cysteine or other essential amino acids, independent of GSH levels. This upregulation did not occur in MEFs lacking GCN2 (general control non-derepressible 2, also known as eIF2α kinase 4) or in cells expressing mutant eIF2α lacking the eIF2α kinase Ser(51) phosphorylation site, indicating that expression of both GCLC and GCLM was mediated by the GCN2/ATF4 stress response pathway. Only the increase in GCLM mRNA level, however, was accompanied by a parallel increase in protein expression, suggesting that the enhanced capacity for GSH synthesis depended largely on increased association of GCLC with its regulatory subunit. Upregulation of both GCLC and GLCM mRNA levels in response to cysteine deprivation was dependent on new protein synthesis, which is consistent with expression of GCLC and GCLM being mediated by proteins whose synthesis depends on activation of the GCN2/ATF4 pathway. Our data suggest that the regulation of GCLC expression may be mediated by changes in the abundance of transcriptional regulators, whereas the regulation of GCLM expression may be mediated by changes in the abundance of mRNA stabilizing or destabilizing proteins. Upregulation of GCLM levels in response to low cysteine levels may serve to protect the cell in the face of a future stress requiring GSH as an antioxidant or conjugating/detoxifying agent.

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.

Mammalian cysteine dioxygenase (CDO) is a mononuclear non-heme iron protein that catalyzes the conversion of cysteine (Cys) to cysteine sulfinic acid by an unclarified mechanism. One structural study revealed that a Cys-persulfenate (or Cys-persulfenic acid) formed in the active site, but quantum mechanical calculations have been used to support arguments that it is not an energetically feasible reaction intermediate. Here, we report a series of high-resolution structures of CDO soaked with Cys at pH values from 4 to 9. Cys binding is minimal at pH≤5 and persulfenate formation is consistently seen at pH values between 5.5 and 7. Also, a structure determined using laboratory-based X-ray diffraction shows that the persulfenate, with an apparent average O-O separation distance of ~1.8Å, is not an artifact of synchrotron radiation. At pH≥8, the active-site iron shifts from 4- to 5-coordinate, and Cys soaks reveal a complex with Cys, but no dioxygen, bound. This 'Cys-only' complex differs in detail from a previously published 'Cys-only' complex, which we reevaluate and conclude is not reliable. The high-resolution structures presented here do not resolve the CDO mechanism but do imply that an iron-bound persulfenate (or persulfenic acid) is energetically accessible in the CDO active site, and that CDO active-site chemistry in the crystals is influenced by protonation/deprotonation events with effective pKa values near ~5.5 and ~7.5 that influence Cys binding and oxygen binding/reactivity, respectively. Furthermore, this work provides reliable ligand-bound models for guiding future mechanistic considerations., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

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.

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.

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.

Cysteine dioxygenase (CDO, EC 1.13.11.20) is a non-heme mononuclear iron enzyme that oxidizes cysteine to cysteinesulfinate. CDO catalyzes the first step in the pathway of taurine synthesis from cysteine as well as the first step in the catabolism of cysteine to pyruvate and sulfate. Previous attempts to purify CDO have been associated with partial or total inactivation of CDO. In an effort to obtain highly purified and active CDO, recombinant rat CDO was heterologously expressed and purified, and its activity profile was characterized. The protein was expressed as a fusion protein bearing a polyhistidine tag to facilitate purification, a thioredoxin tag to improve solubility, and a factor Xa cleavage site to permit removal of the entire N-terminus, leaving only the 200 amino acids inherent to the native protein. A multi-step purification scheme was used to achieve >95% purity of CDO. The approximately 40.3 kDa full-length fusion protein was purified to homogeneity using a three-column scheme, the fusion tag was then removed by digestion with factor Xa, and a final column step was used to purify homogeneous approximately 23 kDa CDO. The purified CDO had high specific activity and kinetic parameters that were similar to those for non-purified rat liver homogenate, including a Vmax of approximately 1880 nmol min-1 mg-1 CDO (kcat=43 min-1) and a Km of 0.45 mM for L-cysteine. The expression and purification of CDO in a stable, highly active form has yielded significant insight into the kinetic properties of this unique thiol dioxygenase.

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.

Hepatic cysteine dioxygenase (CDO) activity is a critical regulator of cellular cysteine concentration and availability of cysteine for anabolic processes and is markedly higher in animals fed diets containing excess sulfur amino acids compared with those fed levels at or below the requirement. Rat hepatocytes responded to a deficiency or excess of cysteine in the culture medium with a decrease or increase in CDO level but no change in CDO mRNA level. The cysteine analog, cysteamine, but not cysteine metabolites or thiol reagents, was also effective in increasing CDO. Inhibitors of the 26S proteasome blocked CDO degradation in cysteine-deficient cells but had little or no effect on CDO concentration in hepatocytes cultured with excess cysteine. High-molecular-mass CDO-ubiquitin conjugates were observed in cells cultured in cysteine-deficient medium, whether or not proteasome inhibitor was present, but these CDO-ubiquitin conjugates were not observed in cells cultured in cysteine-supplemented medium with or without proteasome inhibitor. Similar results were observed for degradation of recombinant CDO expressed in human heptocarcinoma cells cultured in cysteine-deficient or cysteine-supplemented medium. CDO is an example of a mammalian enzyme that is robustly regulated via its substrate, with the presence of substrate blocking the ubiquitination of CDO and, hence, the targeting of CDO for proteasomal degradation. This regulation occurs in primary hepatocytes in a manner that corresponds with changes observed in intact animals.

Two hepatic enzymes, cysteine dioxygenase (CDO) and gamma-glutamylcysteine synthetase (GCS), play important regulatory roles in the response of cysteine metabolism to changes in dietary sulfur amino acid or protein levels. To examine the time-course of changes in CDO and GCS activities, CDO and GCS-catalytic or heavy subunit protein and mRNA levels, and cysteine and glutathione levels, we adapted rats to either a low protein (LP) or high protein (HP) diet, switched them to the opposite diet, and followed these parameters over 6 days. Hepatic CDO activity and amount, but not mRNA level, increased in response to higher protein intake; the t(1/2) of change for CDO activity or protein level was 22 h for rats switched from a LP to a HP diet and 8 h for rats switched from a HP to a LP diet, suggesting that the HP diet decreased turnover of CDO. Hepatic GCS activity, catalytic subunit amount and mRNA level decreased in response to a higher protein intake. GCS catalytic subunit level changed with a similar t(1/2) for both groups, but the change in GCS activity in rats switched from a LP diet to a HP diet was faster (approximately 16h) than for rats switched from a HP to a LP diet (approximately 74h). Hepatic cysteine and glutathione levels reached new steady states within 12 h (LP to HP) or 24 h (HP to LP). CDO activity appeared to be regulated at the level of protein, probably by diminished turnover of CDO in response to higher protein intake or cysteine level, whereas GCS activity appeared to be regulated both at the level of mRNA and activity state in response to the change in cysteine or protein availability. These findings support a role of cysteine concentration as a mediator of its own metabolism, favoring catabolism when cysteine is high and glutathione synthesis when cysteine is low.

Hepatocyte preparations highly enriched in cells from either the periportal or the perivenous zone of the liver acinus were prepared using a digitonin/collagenase perfusion method. Five enzymes of cysteine metabolism were assayed in both periportal and perivenous preparations. The ratios of periportal to perivenous activity were 0.76, 0.60, 0.81, 1.62, and 1.01 for cysteine dioxygenase, cysteinesulfinate decarboxylase, gamma-glutamylcysteine synthetase, cystathionase, and asparate (cysteinesulfinate) aminotransferase, respectively. Only cysteinesulfinate decarboxylase activity was significantly different between periportal and perivenous cells. In incubations with 2 mmol/L [(35)S]cysteine, total cysteine catabolism ([(35)S]taurine plus [(35)S]sulfate) between periportal and perivenous cells was not different, which is consistent with the observation of similar cysteine dioxygenase activity across the hepatic acinus. Consistent with the lower cysteinesulfinate decarboxylase activity in periportal cells, 16% of the total catabolism of [(35)S]cysteine in periportal cells resulted in taurine synthesis compared to 28% in perivenous cells. A lower rate of [(35)S]glutathione synthesis was observed in periportal cells compared to perivenous cells, but gamma-glutamylcysteine synthetase activity was not significantly different between perivenous and periportal cells. Cysteinesulfnate decarboxylase can be added to the list of enzymes whose activities are markedly enriched in perivenous cells.

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.

Conflicting reports in the literature of appropriate assay procedures for measurement of cysteine dioxygenase activity led us to evaluate the procedure for assay of cysteine dioxygenase activity in rat liver preparations. Cysteine dioxygenase activity was largely in the soluble fraction of liver and was stimulated by addition of NAD+ and Fe2+. The pH optimum of the enzyme was 6.1. Addition of an inhibitor of pyridoxal 5-phosphate-dependent enzymes was necessary to prevent rapid removal of the reaction product cysteine sulfinate. Cysteine sulfinate and cysteic acid were separated by anion-exchange HPLC on a polymer-based column with trimethylamino active groups, and the reaction products were quantitated by measurement of 35S radioactivity or by formation and measurement of fluorescent derivatives. This assay of cysteine dioxygenase under optimal conditions provides a physiologically relevant measure of cysteine dioxygenase activity in liver and hepatocytes based on the observation that this activity was highly correlated with the capacity for cysteine catabolism and taurine production by isolated hepatocytes.

The metabolism of cysteine and related compounds was investigated in the isolated perfused hindquarter of the rat. An erythrocyte-based perfusion medium was used; use of a perfluorochemical emulsion, FC-43, resulted in apparent chemical oxidation of cysteine, whereas bovine erythrocytes did not appear to contribute significantly to the metabolism of cysteine. Rat skeletal muscle perfused with L-[35S]cysteine, L-[35S]cystine, L-[35S]cysteinesulfinate, or L-2-oxo-[35S]thiazolidine-4-carboxylate (OTC) for 2 h produced [35S]sulfate and [35S]taurine. In all cases, the partitioning of cysteine or cysteinesulfinate between metabolism to taurine and sulfate was similar, suggesting that cysteine metabolism in hindquarter may occur via formation and catabolism of cysteinesulfinate by either cysteinesulfinate decarboxylase or aspartate (cysteine-sulfinate) aminotransferase. However, the activity of cysteine dioxygenase was extremely low, suggesting that the conversion of cysteine to cysteinesulfinate may have been non-enzymatic.

The uptake and metabolism of L-2-oxo-[35S]thiazolidine-4-carboxylate (OTC) was compared with that of L-[35S]cysteine and L-[35S]methionine in studies with freshly isolated rat hepatocytes, renal cortical tubules and enterocytes. All three 35S-labeled substrates were metabolized to glutathione, inorganic sulfur and taurine by hepatocytes and to inorganic sulfur by renal tubules and enterocytes. The rate of metabolite production from OTC was always less than 30% of that from cysteine or methionine. The transport rate for uptake of [35S]OTC by hepatocytes was less than that observed for uptake of [35S]cysteine or [35S]methionine. The capacity of rat hepatocytes, renal cortical tubules and enterocytes to take up and metabolize OTC is substantially lower than that for uptake and metabolism of cysteine or its normal intracellular precursor, methionine.

Both cysteinesulfinate-independent and cysteinesulfinate-dependent pathways are involved in the catabolism of cyst(e)ine by freshly isolated rat renal cortical tubules. Sulfate and thiosulfate were shown to be the major sulfur-containing products that accumulated in incubations of renal tubules with 1 mmol/L or 25 mmol/L [35S]cyst(e)ine. Thiosulfate is an intermediate in the oxidation of the sulfide produced by the cysteinesulfinate-independent catabolism of cyst(e)ine by desulfhydration pathway(s), whereas sulfate is formed both by further oxidation of thiosulfate and by oxidation of the sulfite formed by the cysteinesulfinate-transamination pathway. Incubation of renal tubules with propargylglycine inhibited gamma-cystathionase activity by 85%, and this resulted in a 46% decrease in sulfate production and a 68% decrease in thiosulfate production when the treated renal tubules were incubated with 1 mmol/L [35S]cyst(e)ine. Addition of 25 mmol/L unlabeled cysteinesulfinate to create a diluting/trapping pool for [35S]cysteinesulfinate formed from [35S]cysteine resulted in a 53% decrease in [35S]sulfate production in incubations with 1 mmol/L cysteine. Thus, some cyst(e)ine catabolism probably occurred by a cysteinesulfinate-dependent pathway. No production of taurine or hypotaurine was detected in incubations with cyst(e)ine. Thus, cysteinesulfinate formed from cysteine was further catabolized primarily to sulfate instead of to taurine and hypotaurine. Most cyst(e)ine catabolism by the epithelial cells of the renal tubule probably can be accounted for by two pathways: 1) the beta-cleavage of cystine catalyzed by lambda-cystathionase and 2) the formation and transamination of cysteinesulfinate catalyzed by cysteine dioxygenase and aspartate aminotransferase.

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.