Overexpression of cysteine dioxygenase reduces intracellular cysteine and glutathione pools in HepG2/C3A cells

Cysteine levels are carefully regulated in mammals to balance metabolic needs against the potential for cytotoxicity. It has been postulated that one of the major regulators of intracellular cysteine levels in mammals is cysteine dioxygenase (CDO). Hepatic expression of this catabolic enzyme increases dramatically in response to increased cysteine availability and may therefore be part of a homeostatic response to shunt excess toxic cysteine to more benign metabolites such as sulfate or taurine. Direct experimental evidence, however, is lacking to support the hypothesis that CDO is capable of altering steady-state intracellular cysteine levels. In this study, we expressed either the wild-type (WT) or a catalytically inactivated mutant (H86A) isoform of CDO in HepG2/C3A cells (which do not express endogenous CDO protein) and cultured them in different concentrations of extracellular cysteine. WT CDO, but not H86A CDO, was capable of reducing intracellular cysteine levels in cells incubated in physiologically relevant concentrations of cysteine. WT CDO also decreased the glutathione pool and potentiated the toxicity of CdCl(2). These results demonstrate that CDO is capable of altering intracellular cysteine levels as well as glutathione levels.     

UHPLC-Orbitrap-Fusion-TMS-Based Metabolomics Study of Phenylpropionamides in the Seed of Cannabis sativa L. against Alzheimer's Disease

Phenylpropionamides in the seed of Cannabis sativa L. (PHS) have a protective effect on neuroinflammation and antioxidant activity. In this study, the UHPLC-Orbitrap-fusion-TMS-based metabolomics approach was used to analyze the serum samples and identify potential biomarkers in Streptozotocin (STZ) induced Alzheimer's disease (AD) rats. The results revealed that primary bile acid biosynthesis and taurine and hypotaurine metabolism were significantly correlated with STZ-induced AD rats. In addition, the key enzymes in these two pathways were verified at the protein level. The levels of cysteine dioxygenase type I (CDO1), cysteine sulfinic acid decarboxylase (CSAD), cysteamine (2-aminoethanethiol) dioxygenase (ADO), 7α-hydroxylase (CYP7A1), and sterol 12α-hydroxylase (CYP8B1) were the key enzymes affecting the two pathways in AD rats compared with the control group (CON). Furthermore, after a high-dose group of phenylpropionamides in the seed of Cannabis sativa L. (PHS−H) was administrated, the levels of CDO1, CSAD, CYP7A1, and CYP8B1 were all callback. These findings demonstrate for the first time that the anti-AD effect of PHS is associated with the regulation of primary bile acid biosynthesis and taurine and hypotaurine metabolism in STZ-induced AD rats.     

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 body's 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.     

Probes of the catalytic site of cysteine dioxygenase

The first major step of cysteine catabolism, the oxidation of cysteine to cysteine sulfinic acid, is catalyzed by cysteine dioxygenase (CDO). In the present work, we utilize recombinant rat liver CDO and cysteine derivatives to elucidate structural parameters involved in substrate recognition and x-ray absorption spectroscopy to probe the interaction of the active site iron center with cysteine. Kinetic studies using cysteine structural analogs show that most are inhibitors and that a terminal functional group bearing a negative charge (e.g. a carboxylate) is required for binding. The substrate-binding site has no stringent restrictions with respect to the size of the amino acid. Lack of the amino or carboxyl groups at the alpha-carbon does not prevent the molecules from interacting with the active site. In fact, cysteamine is shown to be a potent activator of the enzyme without being a substrate. CDO was also rendered inactive upon complexation with the metal-binding inhibitors azide and cyanide. Unlike many non-heme iron dioxygenases that employ alpha-keto acids as cofactors, CDO was shown to be the only dioxygenase known to be inhibited by alpha-ketoglutarate.     

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.     

Heterologous expression, purification, and characterization of recombinant rat cysteine dioxygenase

Cysteine dioxygenase (CDO, EC 1.13.11.20) catalyzes the oxidation of cysteine to cysteine sulfinic acid, which is the first major step in cysteine catabolism in mammalian tissues. Rat liver CDO was cloned and expressed in Escherichia coli as a 26.8-kDa N-terminal fusion protein bearing a polyhistidine tag. Purification by immobilized metal affinity chromatography yielded homogeneous protein, which was catalytically active even in the absence of the secondary protein-A, which has been reported to be essential for activity in partially purified native preparations. As compared with those existing purification protocols for native CDO, the milder conditions used in the isolation of the recombinant CDO allowed a more controlled study of the properties and activity of CDO, clarifying conflicting findings in the literature. Apo-protein was inactive in catalysis and was only activated by iron. Metal analysis of purified recombinant protein indicated that only 10% of the protein contained iron and that the iron was loosely bound to the protein. Kinetic studies showed that the recombinant enzyme displayed a K(m) value of 2.5 +/- 0.4 mm at pH 7.5 and 37 degrees C. The enzyme was shown to be specific for l-cysteine oxidation, whereas homocysteine inhibited CDO activity.     

The ubiquitin-proteasome system is responsible for cysteine-responsive regulation of cysteine dioxygenase concentration in liver

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.     

A putative Fe2+-bound persulfenate intermediate in cysteine dioxygenase

The common reactions of dioxygen, superoxide, and hydroperoxides with thiolates are thought to proceed via persulfenate intermediates, yet these have never been visualized. Here we report a 1.4 A resolution crystal structure of the Fe(2+)-dependent enzyme cysteine dioxygenase (CDO) containing this putative intermediate trapped in its active site pocket. The complex raises the possibility that, distinct from known dioxygenases and proposed CDO mechanisms, the Fe-proximal oxygen atom may be involved in the primary oxidation event yielding a unique three-membered Fe-S-O cyclic intermediate. A nonpolar environment of the distal oxygen would facilitate isomerization of the persulfenate to the sulfinate product.     

Cysteine regulates expression of cysteine dioxygenase and gamma-glutamylcysteine synthetase in cultured rat hepatocytes

Rat hepatocytes cultured for 3 days in basal medium expressed low levels of cysteine dioxygenase (CDO) and high levels of gamma-glutamylcysteine synthetase (GCS). When the medium was supplemented with 2 mmol/l methionine or cysteine, CDO activity and CDO protein increased by >10-fold and CDO mRNA increased by 1.5- or 3.2-fold. In contrast, GCS activity decreased to 51 or 29% of basal, GCS heavy subunit (GCS-HS) protein decreased to 89 or 58% of basal, and GCS mRNA decreased to 79 or 37% of basal for methionine or cysteine supplementation, respectively. Supplementation with cysteine consistently yielded responses of greater magnitude than did supplementation with an equimolar amount of methionine. Addition of propargylglycine to inhibit cystathionine gamma-lyase activity and, hence, cysteine formation from methionine prevented the effects of methionine, but not those of cysteine, on CDO and GCS expression. Addition of buthionine sulfoximine to inhibit GCS, and thus block glutathione synthesis from cysteine, did not alter the ability of methionine or cysteine to increase CDO. GSH concentration was not correlated with changes in either CDO or GCS-HS expression. The effectiveness of cysteine was equivalent to or greater than that of its precursors (S-adenosylmethionine, cystathionine, homocysteine) or metabolites (taurine, sulfate). Taken together, these results suggest that cysteine itself is an important cellular signal for upregulation of CDO and downregulation of GCS.     

Thiol dioxygenases: unique families of cupin proteins

Proteins in the cupin superfamily have a wide range of biological functions in archaea, bacteria and eukaryotes. Although proteins in the cupin superfamily show very low overall sequence similarity, they all contain two short but partially conserved cupin sequence motifs separated by a less conserved intermotif region that varies both in length and amino acid sequence. Furthermore, these proteins all share a common architecture described as a six-stranded β-barrel core, and this canonical cupin or “jelly roll” β-barrel is formed with cupin motif 1, the intermotif region, and cupin motif 2 each forming two of the core six β-strands in the folded protein structure. The recently obtained crystal structures of cysteine dioxygenase (CDO), with contains conserved cupin motifs, show that it has the predicted canonical cupin β-barrel fold. Although there had been no reports of CDO activity in prokaryotes, we identified a number of bacterial cupin proteins of unknown function that share low similarity with mammalian CDO and that conserve many residues in the active-site pocket of CDO. Putative bacterial CDOs predicted to have CDO activity were shown to have similar substrate specificity and kinetic parameters as eukaryotic CDOs. Information gleaned from crystal structures of mammalian CDO along with sequence information for homologs shown to have CDO activity facilitated the identification of a CDO family fingerprint motif. One key feature of the CDO fingerprint motif is that the canonical metal-binding glutamate residue in cupin motif 1 is replaced by a cysteine (in mammalian CDOs) or by a glycine (bacterial CDOs). The recent report that some putative bacterial CDO homologs are actually 3-mercaptopropionate dioxygenases suggests that the CDO family may include proteins with specificities for other thiol substrates. A paralog of CDO in mammals was also identified and shown to be the other mammalian thiol dioxygenase, cysteamine dioxygenase (ADO). A tentative fingerprint motif for ADOs, or DUF1637 family members, is proposed. In ADOs, the conserved glutamate residue in cupin motif 1 is replaced by either glycine or valine. Both ADOs and CDOs appear to represent unique clades within the cupin superfamily.     

Crystal structure of human cysteamine dioxygenase provides a structural rationale for its function as an oxygen sensor

Cysteamine dioxygenase (ADO) plays a vital role in regulating thiol metabolism and preserving oxygen homeostasis in humans by oxidizing the sulfur of cysteamine and N-terminal cysteine-containing proteins to their corresponding sulfinic acids using O₂ as a cosubstrate. However, as the only thiol dioxygenase that processes both small-molecule and protein substrates, how ADO handles diverse substrates of disparate sizes to achieve various reactions is not understood. The knowledge gap is mainly due to the three-dimensional structure not being solved, as ADO cannot be directly compared with other known thiol dioxygenases. Herein, we report the first crystal structure of human ADO at a resolution of 1.78 Å with a nickel-bound metal center. Crystallization was achieved through both metal substitution and C18S/C239S double mutations. The metal center resides in a tunnel close to an entry site flanked by loops. While ADO appears to use extensive flexibility to handle substrates of different sizes, it also employs proline and proline pairs to maintain the core protein structure and to retain the residues critical for catalysis in place. This feature distinguishes ADO from thiol dioxygenases that only oxidize small-molecule substrates, possibly explaining its divergent substrate specificity. Our findings also elucidate the structural basis for ADO functioning as an oxygen sensor by modifying N-degron substrates to transduce responses to hypoxia. Thus, this work fills a gap in structure–function relationships of the thiol dioxygenase family and provides a platform for further mechanistic investigation and therapeutic intervention targeting impaired oxygen sensing.     

Expression, purification, and kinetic characterization of recombinant rat cysteine dioxygenase, a non-heme metalloenzyme necessary for regulation of cellular cysteine levels

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.     

Enhancement of menadione stress tolerance in yeast by accumulation of hypotaurine and taurine: co-expression of cDNA clones, from Cyprinus carpio, for cysteine dioxygenase and cysteine sulfinate decarboxylase in Saccharomyces cerevisiae

Taurine is known to function as a protectant against various stresses in animal cells. In order to utilize taurine as a compatible solute for stress tolerance of yeast, isolation of cDNA clones for genes encoding enzymes involved in biosynthesis of taurine was attempted. Two types of cDNA clones corresponding to genes encoding cysteine dioxygenase (CDO1 and CDO2) and a cDNA clone for cysteine sulfinate decarboxylase (CSD) were isolated from Cyprinus carpio. Deduced amino acid sequences of the two CDOs and that of CSD showed high similarity to those of CDOs and those of CSDs from other organisms, respectively. The coding regions of CDO1, CDO2, and CSD were subcloned into an expression vector, pESC-TRP, for Saccharomyces cerevisiae. Furthermore, to enhance the efficiency of synthesis of taurine in S. cerevisiae, a CDO–CSD fusion was designed and expressed. Expression of CDO and CSD proteins, or the CDO–CSD fusion protein was confirmed by Western blot analysis. HPLC analysis showed that the expression of the proteins led to enhancement of the accumulation level of hypotaurine, a precursor of taurine, rather than taurine. The yeast cells expressing corresponding genes showed tolerance to oxidative stress induced by menadione, but not to freezing–thawing stress.     

Correlating crosslink formation with enzymatic activity in cysteine dioxygenase

Cysteine dioxygenase (CDO) from rat and other mammals exhibits a covalent post-translational modification between the residues C93 and Y157 that is in close proximity to the active site, and whose presence enhances the enzyme's activity. Protein with and without C93-Y157 crosslink migrates as distinct bands in SDS-PAGE, allowing quantification of the relative ratios between the two forms by densitometry of the respective bands. Expression of recombinant rat wild type CDO in Escherichia coli typically produces 40-50% with the C93-Y157 crosslink. A strategy was developed to increase the ratio of the non-crosslinked form in an enzyme preparation of reasonable quantity and purity, allowing direct assessment of the activity of non-crosslinked CDO and mechanism of formation of the crosslink. The presence of ferrous iron and oxygen is a prerequisite for C93-Y157 crosslink formation. Absence of oxygen during protein expression increased the fraction of non-crosslinked CDO, while presence of the metal chelator EDTA had little effect. Metal affinity chromatography was used to enrich non-crosslinked content. Both the enzymatic rate of cysteine oxidation and the amount of cross-linking between C93 and Y157 increased significantly upon exposure of CDO to air/oxygen and substrate cysteine in the presence of iron in a hitherto unreported two-phase process. The instantaneous activity was proportional to the amount of crosslinked enzyme present, demonstrating that the non-crosslinked form has negligible enzymatic activity. The biphasic kinetics suggest the existence of an as yet uncharacterised intermediate in crosslink formation and enzyme activation.     

Regulation of cysteine dioxygenase and gamma-glutamylcysteine synthetase is associated with hepatic cysteine level

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.     

Endogenous generation of sulfur dioxide in rat tissues

While sulfur dioxide (SO₂) has been previously known for its toxicological effects, it is now known to be produced endogenously in mammals from sulfur-containing amino acid l-cysteine. l-cysteine is catalyzed by cysteine dioxygenase (CDO) to l-cysteinesulfinate, which converts to β-sulfinylpyruvate through transamination by aspartate aminotransferase (AAT), and finally spontaneously decomposes to pyruvate and SO₂. The present study explored endogenous SO₂ production, and AAT and CDO distribution in different rat tissue. SO₂ content was highest in stomach, followed by tissues in the right ventricle, left ventricle, cerebral gray matter, pancreas, lung, cerebral white matter, renal medulla, spleen, renal cortex and liver. AAT activity and AAT1 mRNA expression were highest in the left ventricle, while AAT1 protein expression was highest in the right ventricle. AAT2 and CDO mRNA expressions were both highest in liver tissue. AAT2 protein expression was highest in the renal medulla, but CDO protein expression was highest in liver tissue. In all tissues, AAT1 and AAT2 were mainly distributed in the cytoplasm rather than the nucleus. These observed differences among tissues endogenously generating SO₂ and associated enzymes are important in implicating the discovery of SO₂ as a novel endogenous signaling molecule.     

Characterization of the nitrosyl adduct of substrate-bound mouse cysteine dioxygenase by electron paramagnetic resonance: electronic structure of the active site and mechanistic implications

Mammalian cysteine dioxygenase (CDO) is a non-heme iron metalloenzyme that catalyzes the first committed step in oxidative cysteine catabolism. The active site coordination of CDO comprises a mononuclear iron ligated by the Nepsilon atoms of three protein-derived histidines, thus representing a new variant on the 2-histidine-1-carboxylate (2H1C) facial triad motif. Nitric oxide was used as a spectroscopic probe in investigating the order of substrate-O2 binding by EPR spectroscopy. In these experiments, CDO exhibits an ordered binding of l-cysteine prior to NO (and presumably O2) similar to that observed for the 2H1C class of non-heme iron enzymes. Moreover, the CDO active site is essentially unreactive toward NO in the absence of substrate, suggesting an obligate ordered binding of l-cysteine prior to NO. Typically, addition of NO to a mononuclear non-heme iron center results in the formation of an {FeNO}7 (S = 3/2) species characterized by an axial EPR spectrum with gx, gy, and gz values of approximately 4, approximately 4, and approximately 2, respectively. However, upon addition of NO to CDO in the presence of substrate l-cysteine, a low-spin {FeNO}7 (S = 1/2) signal that accounts for approximately 85% of the iron within the enzyme develops. Similar {FeNO}7 (S = 1/2) EPR signals have been observed for a variety of octahedral mononuclear iron-nitrosyl synthetic complexes; however, this type of iron-nitrosyl species is not commonly observed for non-heme iron enzymes. Substitution of l-cysteine with isosteric substrate analogues cysteamine, 3-mercaptopropionic acid, and propane thiol did not produce any analogous {FeNO}7 signals (S = 1/2 or 3/2), thus reflecting the high substrate specificity of the enzyme observed by a number of researchers. The unusual {FeNO}7 (S = 1/2) electronic configuration adopted by the substrate-bound iron-nitrosyl CDO (termed {ES-NO}7) is a result of the bidentate thiol/amine coordination of l-cysteine in the NO-bound CDO active site. DFT computations were performed to further characterize this species. The DFT-predicted geometric parameters for {ES-NO}7 are in good agreement with the crystallographically determined substrate-bound active site configuration of CDO and are consistent with known iron-nitrosyl model complexes. Moreover, the computed EPR parameters (g and A values) are in excellent agreement with experimental results for this CDO species and those obtained from comparable synthetic {FeNO}7 (S = 1/2) iron-nitrosyl complexes.