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Division of Nutritional Sciences, Cornell University, Ithaca, NY
3 To whom correspondence should be addressed. E-mail: mhs6{at}cornell.edu.
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ABSTRACT |
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 TOP ABSTRACT LITERATURE CITED |
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KEY WORDS: • cysteine • cysteine dioxygenase • glutathione • glutamate-cysteine ligase • hypotaurine • taurine • coenzyme A • cysteamine • hydrogen sulfide
The mammalian liver tightly regulates its intracellular free cysteine pool. In rats, for instance, intracellular cysteine is narrowly maintained between 20 and 100 nmol/g even when dietary protein or sulfur amino acid intake is varied from subrequirement to above-requirement levels for this species (1). The effect of diet on plasma and hepatic cysteine levels is illustrated by the data shown in Figure 1. Rats that had been adapted to a high-protein diet and then fed a low-protein diet supplemented with cysteine had, at 6 h after the diet was introduced, a large increase in the portal plasma cysteine concentration but no increase above the fasting value for cysteine in the arterial plasma or in the liver. On the other hand, the plasma cysteine concentration was not significantly decreased, compared with fasting levels, in rats fed a low-protein diet, whereas the hepatic cysteine concentration was markedly decreased. Thus, in rats, the liver allows its own cysteine concentration to vary about 5-fold (from 20 to 100 nmol/g) while regulating cysteine degradation to maintain the plasma cysteine concentration within a 2.5-fold range (between 80 and 200 µmol/L). By keeping cysteine levels within a very narrow range, the liver addresses 2 opposing homeostatic requirements. Cysteine levels must be sufficiently high to meet the needs of protein synthesis and the production of other essential molecules that include glutathione, coenzyme A, taurine, and inorganic sulfur. At the same time, however, cysteine concentrations must also be kept below the threshold of cytotoxicity. The potent toxicity of excess cysteine has been demonstrated in several animal models (2–4), and chronically high levels of cysteine have been closely associated with rheumatoid arthritis (5), Parkinson's disease (6), Alzheimer's disease (6), systemic lupus erythematosus (7), increased risk of cardiovascular disease (8), and adverse pregnancy outcomes in humans (9).
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0.02 to 0.1 mmol/kg, which is substantially below the Km of CDO for cysteine (0.45 mmol/L), so CDO is able to respond to changes in tissue substrate concentration (1,11–13). In addition, the steady-state levels of hepatic CDO protein are exquisitely regulated by the availability of the dietary sulfur amino acids, methionine and cysteine. Hepatic CDO activity is barely detectable in rats fed low-protein (i.e., sulfur amino acid poor) diets but increases by as much as 35-fold in rats fed diets enriched with methionine, cysteine, or protein (14,15). The ability of methionine to stabilize CDO is dependent, however, on its conversion to cysteine. Blocking the synthesis of cysteine from methionine with the transsulfuration inhibitor propargylglycine inhibits the effect of methionine, but not that of cysteine, on CDO levels (16). A comprehensive evaluation of cysteine structural analogs/metabolites has further shown, both in primary rat hepatocyte cultures (17) and in intact rats (18), that intracellular cysteine is the proximate nutrient signal for CDO stability. Cysteine's ability to regulate CDO protein levels is unusual in that it is an exclusively posttranslational phenomenon (14,15,17,19). Studies with primary rat hepatocytes have shown that extracellular cysteine availability directly affects the intracellular cysteine level, which, in turn, alters the ubiquitination and subsequent degradation of CDO protein. In these studies, under conditions where intracellular cysteine levels were low, CDO was rapidly ubiquitinated and degraded by the 26S proteasome system. In contrast, when intracellular cysteine levels were high, the ubiquitination of CDO was markedly attenuated, and the half-life of the protein was significantly prolonged. Ubiquitination of CDO in response to cysteine deprivation is illustrated in Figure 3, and the overall effect of cysteine supplementation of basal medium (0.1 mmol/L methionine, no cysteine) or of addition of a proteasome inhibitor (lactacystin) on intracellular cysteine and CDO levels is shown in Figure 4. The response of CDO turnover to the level of cysteine in the medium was dose-dependent in the range of 0.1 to 1.0 mmol/L of cysteine in fresh medium, as shown in Figure 5.
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37 nmol/g, rats fed the low-protein diet that were injected with PS1 had hepatic hypotaurine levels of 680 nmol/g at 3.5 h after the injection. Although it has been very well established that cysteine regulates CDO turnover through the ubiquitin–26S proteasome pathway, precisely how the signal for degradation is relayed is not clear. We have shown that the signal for degradation does not appear to involve a simple redox sensor; incubation of hepatocytes with reducing agents such as ascorbic acid, hydrogen sulfide, dithiothreitol, or ß-mercaptoethanol did not prevent the degradation of CDO in cells cultured in a cysteine-deficient medium (18). Nor does it appear that the degradation signal is tied to the presence or absence of substrate in the CDO active site. Cysteamine, which is a close structural analog of cysteine but is neither a substrate for CDO nor a competitive inhibitor of its activity, was capable of stabilizing CDO in hepatocytes cultured in a cysteine-free medium (18).
Regulation of cysteine utilization for glutathione synthesis by complex regulation of glutamate-cysteine ligase: differential expression of subunits and the effect of heterodimer formation on kcat.
Glutathione synthesis occurs in virtually all cells, but the liver plays a particular role in that it is the tissue that takes up and removes the bulk of the dietary cysteine in the portal blood, mainly by converting it to GSH, which it releases into the circulation (10). Tissue GSH levels become depleted at sulfur amino acid intakes that are marginal but adequate for protein synthesis, demonstrating that protein synthesis has a higher priority for cysteine than does GSH synthesis (12,19). The normal turnover of GSH in human adults has been estimated to be 40 mmol per day, which is slightly greater than estimates of the magnitude of cysteine turnover in the body protein pool (20–24). Although GSH turnover returns cysteine to the cysteine pool, and oxidized glutathione can be reduced back to GSH, some cysteine is irreversibly lost through excretion of glutathione or its metabolites (e.g., mercapturic acids).
Glutamate-cysteine ligase (GCL, also known as 
-glutamylcysteine synthetase, EC 6.3.2.2) catalyzes the first step in glutathione synthesis and plays an important role in regulating the flux of cysteine to GSH. The mammalian GCL holoenzyme consists of an 73 kDa catalytic subunit (GCLC) and an 31 kDa modifier subunit (GCLM), which are encoded by separate genes. In vitro, it has been shown that the GCLC subunit exhibits catalytic activity and feedback inhibition by GSH, whereas the GCLM modifier subunit has no enzymatic activity. However, the association of GCLM with GCLC alters the kinetics of the reaction, lowering the Km for glutamate and ATP, increasing the Ki for GSH, and increasing the turnover rate (kcat), but not altering the Km for cysteine (25–27).
Short-term regulation of GSH production by GCL occurs mainly via the availability of cysteine, the limiting substrate, and perhaps by feedback inhibition of GCL by GSH. The Km of rat liver GCL for cysteine is 0.1 mmol/L, near the upper end of typical cellular cysteine concentrations, and the rate of GSH synthesis is extremely sensitive to changes in the cellular cysteine level. Not only is GSH synthesis highly dependent on the cellular cysteine concentration, but normal GSH turnover plays a crucial role in maintenance of cellular cysteine levels. Oral administration of a source of cysteine (N-acetylcysteine) reversed or prevented the abnormalities associated with -glutamyl transpeptidase deficiency and hence impaired GSH breakdown in mice (28,29). Furthermore, although depletion of GCLC was lethal in the mouse embryo, supplementation of GCLC-deficient cell lines with N-acetylcysteine allowed these cells to grow indefinitely, whereas supplementation of the cells with dithiothreitol, ascorbic acid, 
-tocopherol, or butylated hydroxytoluene did not rescue the cells from rapid death (30).
Since the initial demonstration that GCL is a heterodimer and the initial characterization of kinetic parameters of the purified enzymes (25,31), it has generally been assumed that the monomer of GCLC would be essentially inactive in vivo and that the holoenzyme would be the major species present in cells (32,33). However, recent measurements of GCL kinetics and of GCLM/GCLC molar ratios yielded the surprising finding that the GCLC monomer is the major species normally present in tissues of intact animals (26). This observation opened the door for exploration of the role of GCLM expression in the regulation of GCL activity.
Both in rat liver and in HepG2 cells exposed to low or marginal cysteine levels, we observe both an increase in the apparent activity state of GCLC and a greater increase in expression of GCLM than of GCLC (11,14–16,26,34). The increased expression of GCLM gives rise to holoenzyme formation, which is associated with an increase in activity state or kcat. For example, in a study of rats switched from a high-protein diet (400 g casein/kg) to a low-protein diet (100 g casein/kg), hepatic GCL activity increased gradually over a 6-d period to reach 2.9 times the initial activity level (26). As summarized in Figure 6, this increase in activity was associated with a 30% increase in the level of GCLC and an 80% increase in the level of GCLM, which resulted in an increase in the molar GCLM/GCLC ratio from 0.23 to 0.34 (1.5 times the initial value). Along with this increase in the GCLM/GCLC molar ratio, the kcat for hepatic GCLC increased from 100 ± 13 nmol·min–1·nmol–1 GCLC (mean ± SEM) to 222 ± 16 nmol·min–1·nmol–1 GCLC. The 30% increase in the absolute level of GCLC, corrected for the 120% increase in the kcat, completely accounted for the 190% higher GCL activity in the liver of rats adapted to the low-protein diet than in liver of rats adapted to the high-protein diet (i.e., 1.3 x 2.2 = 2.9). We also found that increases in GCLC and GCLM subunit levels in rat liver were accompanied by parallel changes in the mRNA levels for GCLC and GCLM, indicating that regulation occurred at the level of mRNA.
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Utilization of cysteine for coenzyme A synthesis: new insights from the roles of pantothenate kinase and pantetheinase in the pathway. Although the pathway for coenzyme A synthesis is well established, the rate of coenzyme A turnover and the extent of cysteine consumption for coenzyme A turnover have not been quantified. The flux-generating step in coenzyme A biosynthesis is the first step in the pathway and is catalyzed by pantothenate kinase. Cysteine is condensed with pantothenate in the second step of the coenzyme A synthesis pathway to form 4'-phosphopantothenoylcysteine, and the cysteine moiety is decarboxylated in the third step of the pathway to form the cysteamine, or ß-mercaptoethylamine, moiety of coenzyme A. The rate of coenzyme A synthesis is determined largely by the regulated pantothenate kinase step, which is highly regulated in response to factors that favor lipid oxidation. Multiple isoforms of mammalian pantothenate kinase (PanK) are encoded by 4 genes in humans and in mice, and the regulatory properties of the various pantothenate kinase isoforms allow the robust control of coenzyme A biosynthesis by coenzyme A and its thioesters (39,40).
The human hereditary disorder pantothenate kinase-associated neurodegeneration (PKAN) has been associated with an accumulation of cysteine in the globus pallidus of the brain (41). Patients with PKAN show a pathological accumulation of iron in the basal ganglia and suffer from a gradual and steady deterioration of movement, speech, and cognition. The recent mapping of this disorder to mutations in the human PanK2 gene (42) suggested that impairment in coenzyme A synthesis could lead to cysteine accumulation in some tissues. Because PanK2 protein is widely expressed in tissues and is localized in the mitochondria, PKAN is thought to diminish mitochondrial function and adversely impact the globus pallidus and the retina, which are tissues with high metabolic requirements that are subject to oxidative damage. Iron accumulation in the brain is presumably caused by the lack of PanK2, which lowers the levels of 4'-phosphopantothenic acid and leads to the buildup of cysteine, which effectively binds iron. Cysteine is cytotoxic and, in the presence of iron, undergoes autooxidation, resulting in free radical formation. Free cysteine also enhances iron-induced lipid peroxidation. Thus, cysteine cytotoxicity as well as oxidative damage in the globus pallidus may contribute to the pathology of PKAN (42). From a metabolic point of view, the accumulation of cysteine suggests that its utilization for coenzyme A synthesis is important for regulation of its concentration in some tissues.
Even less is known about the regulation of coenzyme A degradation. Coenzyme A degradation involves sequential degradation of coenzyme A to dephospho-CoA + Pi, 4'-phosphopantetheine + AMP, and then pantetheine + Pi. In the final step of the degradation pathway, pantetheine is degraded to pantothenic acid and cysteamine (ß-mercaptoethylamine) by an enzyme known as pantetheinase. Cysteamine can function as an antioxidant as well as a precursor for taurine biosynthesis. Little is known about the rate of cysteamine formation in mammalian tissues, but it is known that cysteamine can be converted to hypotaurine and, hence, to taurine.
Pitari et al. (43) recently reported that vanin-1–null mice were deficient in membrane-bound pantetheinase in liver and kidney and had negligible levels of cysteamine in their tissues. Vanin proteins were recently identified as pantetheinases on the basis of sequence similarity with pig pantetheinase (44). Vanin proteins are encoded by 2 genes in mice (vanin-1 and -3) and 3 in humans (vanin-1, -2, and -3) (45–48). The unanticipated observation of pantetheinase deficiency in the vanin-1–null mouse model may facilitate efforts to evaluate the quantitative significance of coenzyme A turnover in vivo. If further work confirms a substantial rate of pantetheine formation and hydrolysis in mammalian tissues, this would imply a substantial rate of coenzyme A turnover, leading to a substantial pool of cysteamine for taurine biosynthesis. In this regard, we have observed higher plasma cysteamine concentrations in rats fed a high-protein (low-carbohydrate) diet (13 ± 2 µmol/L, mean ± SD) than in rats fed a low-protein (high-carbohydrate) diet (2.3 ± 0.7 µmol/L) and also in rats that had been fasted overnight (23 ± 4 µmol/L) compared with rats in an absorptive state (13 ± 2 µmol/L).
Pathways of taurine synthesis: cysteinesulfinate- and cysteamine-dependent pathways. The pathways for taurine synthesis from cysteine are shown in Figure 7. The relative contribution of the cysteinesulfinate-dependent pathway versus the cysteamine-dependent pathway to net taurine production is not clear, largely because the magnitude of flux through the cysteamine pool has not been assessed. Several research groups, including our own laboratory, have focused their efforts on the cysteinesulfinate pathway, and we have shown that flux through this pathway is highly responsive to cysteine concentration (49–53). Regulation of taurine biosynthesis via this pathway is mediated principally at the level of cysteine dioxygenase concentration, which is directly controlled by substrate concentration (17,18). Studies with isolated hepatocytes clearly show that changes in cysteine dioxygenase activity play a dominant role in determining the rates of both taurine and sulfate formation (50–52).
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The high flux of cysteine through the cysteinesulfinate pathway under conditions of excess cysteine availability does not necessarily imply that the cysteamine pathway is a negligible contributor to taurine synthesis. On the contrary, there is ample indirect evidence for the synthesis of taurine via this route in the central nervous system and certain other tissues that express very low levels of cysteine dioxygenase and thus are unlikely to have substantial cysteine 
taurine flux through the cysteinesulfinate pathway (57). Efforts to evaluate flux through the cysteamine pathway have been limited by incomplete data on the rate of coenzyme A turnover (58), by the technical difficulty in measuring cysteamine (59,60), and by the lack of definitive identification of cysteamine dioxygenase, the enzyme responsible for oxidation of cysteamine to hypotaurine (60).
We recently demonstrated that intact rats have a relatively large capacity for conversion of cysteamine to hypotaurine. Rats fed a basal low-protein diet (100 g casein/kg diet) supplemented with cysteamine or an equimolar amount of cysteine had markedly elevated levels of hypotaurine in liver, kidney, and brain at 6 and 10 h after introduction of the supplemented diet. As shown in Figure 8, tissue hypotaurine levels were higher in rats fed a diet supplemented with cysteine than in those fed a diet supplemented with an equimolar amount of cysteamine. This is consistent with cysteine being more readily converted to hypotaurine as a result of cysteine dioxygenase and cysteinesulfinate decarboxylase activities. Nevertheless, the increase (above basal, at 6 h) in hypotaurine level in liver and kidney of rats given supplemental cysteamine was 42 and 52% as much, respectively, as that observed in rats given an equimolar amount of supplemental cysteine, demonstrating that cysteamine is a good precursor of hypotaurine in vivo. This is even more striking because tissue cysteamine concentrations were not increased as much by cysteamine supplementation as cysteine concentrations were increased by cysteine supplementation. Supplementation of the diet with cysteine had no effect on tissue cysteamine concentrations, and supplementation of the diet with cysteamine had no effect on tissue cysteine concentrations. Thus, cysteamine can be converted to hypotaurine at a physiologically significant rate. Depending on the rate of cysteamine production via coenzyme A turnover, cysteamine could be a quantitatively important precursor of taurine.
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-lyase to yield pyruvate, ammonia, and thiocysteine, followed by further reaction of thiocysteine to release sulfide, 2) the transamination of cysteine or cystine by aminotransferases to yield 3-mercaptopyruvate, which is further metabolized by mercaptopyruvate sulfurtransferase to release or transfer the sulfur, and 3) the replacement of the thiol group of cysteine with a variety of thiol compounds to form the corresponding thioether in a reaction catalyzed by cystathionine ß-synthase (61,62). H2S is a potential product of each of these desulfuration pathways. The reduced sulfur may be used in synthesis of molecules requiring a source of reduced sulfur, or it may be oxidized to thiosulfate (inner sulfur atom), sulfite, and finally sulfate. The capacity of cysteine desulfhydration pathways is low and minimally responsive to increased intakes of sulfur amino acids (49,63–65), but these pathways may provide a constitutive pathway for production of reduced sulfur. (In contrast, the cysteinesulfinate-dependent catabolic pathways provide a robust and responsive system for removal of excess cysteine without production of toxic H2S.)
A series of recent studies offer evidence that regulated production of H2S has important physiological functions and further document the role of cystathionine ß-synthase and cystathionine -lyase in H2S production from cysteine. Kimura and co-workers (66,67) demonstrated that the brain produces endogenous H2S from cysteine by cystathionine ß-synthase-dependent desulfuration (68). Observations on patients with Alzheimer's disease also are consistent with a role of cystathionine ß-synthase in H2S production. Eto et al. (69) found that levels of H2S were severely decreased in the brains of Alzheimer's disease patients compared with the brains of age-matched control individuals. Brains of patients with Alzheimer's disease also had reduced cystathionine ß-synthase activity, elevated homocysteine, and a reduced level of S-adenosylmethionine, an activator of cystathionine ß-synthase, and these patients also had elevated plasma homocysteine levels (70). In glutaminergic neurons, the production of H2S by cystathionine ß-synthase enhances N-methyl-D-aspartate (NMDA) receptor-mediated currents (66). NMDA receptor modulation may involve the activation of adenylate cyclase, activation of protein kinase A, and phosphorylation of NMDA receptor subunits (71).
H2S may also play a role as a smooth muscle relaxant. Hosoki et al. (67) reported that cystathionine -lyase mRNA is expressed in the ileum, portal vein, and thoracic aorta, tissues that produce H2S. Inhibitor studies suggested that the production of H2S in portal vein and thoracic aorta was catalyzed by cystathionine -lyase, whereas that in ileum was catalyzed by both cystathionine -lyase and cystathionine ß-synthase. Relaxation of rat aortic tissues, rabbit ileum, and rabbit vas deferens in response to H2S occurred in a dose-related manner (72,73). Additionally, sodium hydrosulfide produced significant dose-dependent decreases in uterine spontaneous contractility (74). Of the amino acids tested, only L-cysteine produced a significant reduction in spontaneous contractility at a dose of 1 mmol/L.
The mechanism by which H2S brings about smooth muscle relaxation is not fully understood. Zhao et al. (75) demonstrated that H2S decreased blood pressure of rats and relaxed aortic tissues by directly opening KATP channels in vascular smooth muscle cells. Cystathionine -lyase was expressed in vascular smooth muscle cells but not in endothelial cells. The vasorelaxant property of H2S on rat aortic tissues was attenuated by Ca2+-dependent K channel blockers or by omission of Ca2+ (67). The action of H2S did not require nitric oxide (NO), but low concentrations of H2S acted synergistically to enhance NO-induced smooth muscle relaxation (75). Sodium nitroprusside, an NO donor, increased cystathionine -lyase expression and stimulated cystathionine -lyase desulfhydration activity in rat aortic and rat lung tissues (67). Thus, the synergistic effects of H2S and NO may be partially explained by NO induction of H2S production.
Summary and relation of cysteine metabolism to amino acid supplement safety. The mammalian liver tightly regulates its free cysteine pool, and intracellular cysteine in rat liver is maintained between 20 and 100 nmol/g even when sulfur amino acid intakes are deficient or excessive. By keeping cysteine levels within a narrow range and by regulating the synthesis of glutathione, which serves as a reservoir of cysteine, the liver addresses both the need to have adequate cysteine to support normal metabolism and the need to keep cysteine levels below the threshold of toxicity. Elevated tissue cysteine levels should be avoided because they may lead to autooxidation of cysteine to form cystine and ROS, oxidation of protein thiol groups, neurotoxicity mediated by NMDA-type glutamate receptors or membrane cystine/glutamate exchanger activity, or excess production of H2S via desulfhydration reactions.
Cysteine catabolism is tightly regulated via regulation of CDO levels in the liver, with the turnover of CDO being dramatically decreased when intracellular cysteine levels increase. This occurs in response to changes in the intracellular cysteine concentration via changes in the rate of CDO ubiquitination and, hence, degradation. Because the response of hepatic CDO to an increase in cysteine or methionine load is rapid (i.e., <24 h to reach new steady-state in rats) and large (i.e., >30-fold increase in CDO protein in rats), the liver provides a substantial safeguard against intake of excess cysteine via the oral dietary route, particularly if dietary changes are made gradually. Individuals with impaired hepatic metabolism, or possible lack of functional CDO, will be at greater risk of excess cysteine intake. Furthermore, it is possible that tissue or plasma hypotaurine levels may serve as a useful biomarker for both insufficient and excess cysteine levels in vivo because our studies with rats indicate that hypotaurine levels are very sensitive to both CDO protein level and to the tissue concentration of cysteine.
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FOOTNOTES |
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2 The work reported here was supported by National Institutes of Health Grants DK-056649 and DK- 0664303.
4 Abbreviations used: CDO, cysteine dioxygenase; CSD, cysteinesulfinate decarboxylase; GCL, glutamate-cysteine ligase; GCLC, GCL catalytic subunit; GCLM, GCL modifier subunit; GSH, glutathione; PKAN, pantothenate kinase-associated neurodegeneration.
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LITERATURE CITED |
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0.05) (JE Dominy, RM Coloso, J-I Lee, and MH Stipanuk, unpublished data).
