Progress 10/01/08 to 09/30/09
Outputs
OUTPUTS: The following projects have been conducted: 1. Using cystallographic studies, we showed that substrate-soaked cysteine dioxygenase (CDO) had a putative Fe2+-bound cysteine persulfenate complex in the active site, which suggests that the oxidation of cysteine to cysteinesulfinate by CDO may proceed through this novel persulfenate intermediate whereby the iron-proximal oxygen atom is involved in the primary oxidation event. Other studies support the conclusion that the cysteine-persulfenate complex was identified correctly and that it is not an irreversibly inactivated complex. 2. To characterize the phenotype of CDO-knockout or CDO-deficient mice, we have successfully generated germ-line CDO knockout mice (CDO-/-) and liver-specific CDO knockdown mice by crossing our CDOflox/flox mice with Cre recombinase transgenic mice (CMV-Cre and Alb-Cre mice, respectively). CDO null mice are born to heterozygous parents (CDO+/-) in the expected Mendelian ratios. The CDO null males are infertile whereas the CDO null females, when mated with a wildtype male, conceive and carry pups to term. The pregnant females had extreme difficulty with parturition, however, with most pups being undelivered and the dams having to be euthanized. CDO null offspring, both male and female, exhibit a severe postnatal growth deficit (~35% lower body weight at 3-5 weeks compared to wildtype littermates). Null mice also exhibit a high rate of postnatal mortality with mortality being greater in null females (~40%) than in null males (~15%).CDO null mice exhibit very low hypotaurine/taurine concentrations in plasma and tissues. Their plasma sulfate and cysteine levels are slightly elevated compared to those of wildtype littermates. CDO null mice exhibit signs of connective tissue and skeletal abnormalities, especially joint hyperlaxity (hypermobility) and wry nose (deviation of the rostral maxilla, perhaps due to chondrodysplasia in the nasal septal cartilage).The dramatic reduction of plasma and tissue hypotaurine/taurine levels in CDO-/- mice indicates that the CDO-dependent pathway of taurine production is the major source of taurine in wildtype mice fed the commercial rodent diet. This clearly indicates that the coenzyme A - cysteamine route of taurine production is quantitatively minor route, although it is possible that this alternative route is physiologically important when the CDO pathway is disrupted. PARTICIPANTS: Not relevant to this project. TARGET AUDIENCES: Not relevant to this project. PROJECT MODIFICATIONS: Not relevant to this project.
Impacts
Results strongly support the formation of an enzyme-bound cysteine-persulfenate intermediate in the catalysis of cysteine dioxygenation by cysteine dioxygenase (CDO). This finding provides new insights into sulfur chemistry and to the reaction chemistry to the thiol dioxygenase families. CDO null offspring are characterized by a postnatal growth deficit; a high level of postnatal mortality that is higher for females than males; hyperlaxity of the joints; wry nose (deviation of the rostral maxilla, perhaps due to chondrodysplasia in the nasal septal cartilage); and very low tissue taurine levels. This model contributes addition insights into the genetic basis of connective tissue disorders. Mutations in the cysteine dioxygenase gene and abnormalities in cysteine metabolism have been associated with more severe, rapidly progressing, and early-onset rheumatoid arthritis in clinical populations. Because some of the metabolic abnormalities arising from loss-of-function mutations in cysteine dioxygenase can be prevented by dietary manipulations, our results could lead to nutritional therapies for individuals with connective tissue diseases.
Publications
- No publications reported this period
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Progress 10/01/07 to 09/30/08
Outputs
OUTPUTS: The following research studies have been conducted on this project: (1) Investigation of the regulation of cysteine dioxygenase, cysteinesulfinate decarboxylase and cysteamine dioxygenase expression in adipose tissue and adipocytes. (2) Elucidation of the mechanism of cysteine dioxygenase protein cofactor (cys-tyr thioether) formation and its effect on enzyme activity. (3) Study of intermediates and reaction mechanism for cysteine dioxygenase by x-ray crystallographic investigations. (4) Investigation of the role of the integrated stress response mediated by phosphorylation of eukaryotic initiation factor 2alpha in response to cysteine deprivation or a low protein diet on sulfur amino acid metabolism. (5) Microarray studies of differential gene expression in response to amino acid deprivation. Results have been disseminated by presentation of seminars, papers at national scientific meetings, and research publications. (6) Generation of a cysteine dioxygenase knockout mouse. PARTICIPANTS: Not relevant to this project. TARGET AUDIENCES: Not relevant to this project. PROJECT MODIFICATIONS: Not relevant to this project.
Impacts
Cysteine dioxygenase plays a crucial role in regulating cellular thiol levels, and cysteine dioxygenase and cysteamine dioxygenase are key enzymes in the two pathways for endogenous taurine biosynthesis. Cysteine dioxygenase activity is regulated in a cysteine-responsive manner via the ubiquitin-proteasome pathway of protein degradation and also via induction of cysteinyl-tyrosine cofactor formation as a consequence of catalytic turnover of substrate. This regulation occurs in both hepatocytes and adipocytes of intact rats and mice. Cysteine dioxygenase is the first example we know of where an enzyme involved in intermediary metabolism has been shown to be upregulated by its substrate via a block in ubiquitylation of the enzyme and also the first example in which substrate directly increases the catalytic efficiency of an enzyme via induction of protein-derived cofactor synthesis. Understanding the mechanism of the regulation of cysteine dioxygenase has the potential to improve the nutrition of both individuals in both health and disease. In addition to the robust downregulation of cysteine dioxygenase in response to dietary deficiency of cysteine or total sulfur amino acids, low cysteine levels dramatically induce the activation of the amino acid deprivation/integrated stress response in liver of rats or in hepatoma cells in culture. The modulatory subunit of glutamate-cysteine ligase, cysteinyl-tRNA synthetase, cystathionase, and cysteine and cystine transporters may be among the genes that are upregulated by the integrated stress response. All of these responses serve to increase cysteine uptake and utilization by cells, while at the same time minimizing the catabolism of cysteine, when cysteine, or its precursor methionine, is limiting.
Publications
- Ueki, I. and Stipanuk, M.H. (2009) 3T3-L1 adipocytes and rat adipose tissue have a high capacity for taurine synthesis by the cysteine dioxygenase/cysteinesulfinate decarboxylase and cysteamine dioxygenase pathways. J. Nutr.(Dec 23 Epub), in press.
- Stipanuk, M.H., Ueki, I., Dominy, J.E. Jr., Simmons, C.R., and Hirschberger, L.L. (2008) Cysteine dioxygenase: a robust system for regulation of cellular cysteine levels. Amino Acids (Nov 15 Epub), in press. Stipanuk, M.H., Dominy, J.E., Jr., Ueki, I., and Hirschberger, L.L. (2008) Measurement of cysteine dioxygenase activity and protein abundance. Curr. Prot. Toxicol. 6.15.1-6.15.25.
- Simmons, C.R., Krishnamoorthy, K., Granett, S.L., Schuller, D.J., Dominy, J.E. Jr., Begley, T.P., Stipanuk, M.H., and Karplus, P.A. (2008) A putative Fe2+-bound persulfenate intermediate in cysteine dioxygenase. Biochemistry 47:11390-11392.
- Dominy, J.E. Jr., Hwang, J., Guo, S., Hirschberger, L.L., Zhang, S., and Stipanuk, M.H. (2008) Synthesis of amino acid cofactor in cysteine dioxygenase is regulated by substrate and represents a novel post-translational regulation of activity. J. Biol. Chem. 283:12188-12201.
- Lee, J-I., Dominy, J.E., Jr., Sikalidis, A. K., Hirschberger, L.L., Wang,W., and Stipanuk, M.H. (2008) HepG2/C3A cells respond to cysteine-deprivation by induction of the amino acid deprivation/integrated stress response pathway. Physiol. Genomics 33:218-229.
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Progress 10/01/06 to 09/30/07
Outputs
The following is research studies have been conducted on this project: (1) Investigation of the regulation of cysteine dioxygenase expression in adipose tissue during differentiation. (2) Investigation of the regulation of cysteine dioxygenase and cysteinesulfinate decarboxylation expression and taurine synthesis in mammary gland during pregnancy and lactation. (3) Histological investigation of the tissue and cell-type specific distribution of cysteine dioxygenase expression in the mouse. (4) Elucidation of the mechanism of cysteine dioxygenase protein cofactor (cys-tyr thioether) formation and its effect on enzyme activity, and further x-ray crystallographic investigations of enzyme mechanism for cysteine dioxygenase. (5) Identification of gene for cysteamine dioxygenase;expression and characterization of cysteamine dioxygenase, the second mammalian thiol dioxygenase. (6) Demonstration that cysteine dioxygenase levels can regulate intracellular cysteine and glutathione
levels and the ability of the cell to respond to chemical stress. (7) Investigation of the role of the integrated stress response mediated by phosphorylation of eukaryotic initiation factor 2alpha in response to cysteine deprivation or a low protein diet on sulfur amino acid metabolism. (8) Microarray studies of differential gene expression in response to amino acid deprivation. Results have been disseminated by presentation of seminars, papers at national scientific meetings, and research publications.
Impacts
Cysteine dioxygenase plays a crucial role in regulating cellular thiol levels, and cysteine dioxygenase and cysteamine dioxygenase are key enzymes in the two pathways for endogenous taurine biosynthesis. Cysteine dioxygenase activity is regulated in a cysteine-responsive manner via the ubiquitin-proteasome pathway of protein degradation and also via induction of cysteinyl-tyrosine cofactor formation as a consequence of catalytic turnover of substrate. This is the first example we know of where an enzyme involved in intermediary metabolism has been shown to be upregulated by its substrate via a block in ubiquitylation of the enzyme and also the first example in which substrate directly increases the catalytic efficiency of an enzyme via induction of cofactor synthesis. The cell type-specific distribution of cysteine dioxygenase in certain nonhepatic tissues suggests other possible roles for cysteine dioxygenase, such as mediating sulfate production for glycosaminoglycan
synthesis. Understanding the mechanism of the regulation of cysteine dioxygenase has the potential to improve the nutrition of both individuals in both health and disease. In addition to the robust downregulation of cysteine dioxygenase in response to dietary deficiency of cysteine or total sulfur amino acids, low cysteine levels dramatically induce the activation of the amino acid deprivation/integrated stress response in liver of rats or in hepatoma cells in culture. The modulatory subunit of glutamate-cysteine ligase, cysteinyl-tRNA synthetase, cystathionase, and cysteine and cystine transporters may be among the genes that are upregulated by the integrated stress response. All of these responses serve to increase cysteine uptake and utilization by cells, while at the same time minimizing the catabolism of cysteine, when cysteine, or its precursor methionine, is limiting.
Publications
- Dominy, J.E. Jr., Hwang, J. and Stipanuk, M.H. 2007. Overexpression of cysteine dioxygenase reduces intracellular cysteine and glutathione pools in HepG2/C3A cells. Am. J. Physiol. Endocrinol. Metab. 293(1):E62-E69.
- Ueki, I. and Stipanuk, M.H. 2007. Enzymes of the taurine biosynthetic pathway are expressed in rat mammary gland. J. Nutr. 137(8):1887-1894.
- Stipanuk, M.H. 2007. The keto acid of methionine is a safe and efficacious substitute for dietary L-methionine: the answer from chick bioassays. J. Nutr. 137(8):1844-1845.
- Dominy, J.E. Jr., Simmons, C.R., Hirschberger, L.L., Hwang, J., Coloso, R.M., and Stipanuk, M.H. 2007. Discovery and characterization of a second mammalian thiol dioxygenase, cysteamine dioxygenase. J. Biol. Chem. 282(35):25189-25198.
- Stipanuk, M.H. 2007. Leucine and protein synthesis: mTOR and beyond. Nutr. Rev. 65(3):122-129.
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Progress 01/01/06 to 12/31/06
Outputs
The following is a list of accomplishments on this project: (1) Purification and kinetic analysis of homogenous cysteine dioxygenase, an iron metalloenzyme. (2) Determination of crystal structure of cysteine dioxygenase, whose structure establishes it as a prototype for a new sub-family of cupin proteins. (3) Demonstration that cysteine is the physiological mediator of cysteine dioxygenase stabilization, via inhibition of ubiquitination/degradation of the protein, in intact rats. To our knowledge, this 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. (4) Demonstration that accumulation of cysteine dioxygenase in rats due to inhibition of the 26S proteasome results in accumulation of polyubiquitinated cysteine dioxygenase, an increase in cysteine dioxygenase protein, and an increase in tissue and plasma hypotaurine levels (indicating the
role of cysteine dioxygenase in sulfoxidation flux). (5) Demonstration that expression of cysteine dioxygenase in hepatoma cells (which do not express endogenous cysteine dioxygenase) results in downregulation of intracellular cysteine levels. (6) Demonstration of abundant expression of cysteine dioxygenase in non-hepatic tissues including adipose tissue and mammary gland. (7) Demonstration that the regulation of glutathione synthesis in response to dietary protein/sulfur amino acid intake is regulated via the differential expression of the catalytic and modifier subunits of glutamate-cysteine ligase. (8) Demonstration that most glutamate-cysteine ligase in liver in vivo exists mainly as the catalytic subunit monomer, and that an increase in expression of the modifier subunit in response to protein/sulfur amino acid deprivation results in increased holoenzyme/heterodimer formation with a large increase in catalytic rate (kcat). (9) Identification of gene for cysteamine
dioxygenase;expression and characterization of cysteamine dioxygenase, the second mammalian thiol dioxygenase.
Impacts
Cysteine dioxygenase plays a crucial role in regulating cellular thiol levels. In is now known that thiol redox state is involved in regulation of the function of a number of cellular proteins including transcription factors. Recent evidence indicates that production of H2S from cysteine has important physiological roles in nervous tissue and smooth muscle. Regulation of cysteine levels is largely accomplished by regulation of hepatic cysteine dioxygenase activity. Cysteine dioxygenase activity is regulated in a cysteine-responsive manner via the ubiquitin-proteasome pathway of protein degradation. This is the first example we know of where an enzyme involved in intermediary metabolism has been shown to be upregulated by its substrate via a block in ubiquitylation of the enzyme. Understanding the mechanism of the regulation of cysteine dioxygenase has the potential to improve the nutrition of both individuals in both health and disease.
Publications
- Coloso, R.M., Hirschberger, L.L. Dominy, J.E. Jr., Lee, J-I. and Stipanuk, M.H. 2006. Cysteamine dioxygenase: evidence for the physiological conversion of cysteamine to hypotaurine in rat and mouse tissues. Adv. Exp. Biol. 583:25-36.
- Simmons, C.R. Hirschberger, L.L., Machi, M.S. and Stipanuk, M.H. 2006. Expression, purification, and kinetic characterization of recombinant rat cysteine dioxygenase, a non-heme metalloenzyme necessary for regulation of cellular cysteine levels. Protein Expr. Purif. 47:74-81.
- Dominy, J.E., Jr., Hirschberger, L.L., Coloso, R.M. and Stipanuk, M.H. 2006. In vivo regulation of cysteine dioxygenase via the ubiquitin-26S proteasome system. Adv. Exp. Biol. Med. 583:37-47.
- Stipanuk, M.H. and Dominy, J.E. Jr. 2006. Surprising insights that aren't so surprising in the modeling of sulfur amino acid metabolism. Amino Acids 30:251-256.
- Simmons, C.R., Liu, Q., Huang, Q., Hao, Q., Begley, T.P., Karplus, P.A. and Stipanuk, M.H. 2006. Crystal structure of mammalian cysteine dioxygenase: A novel mononuclear iron center for cysteine thiol oxidation. J Biol Chem. 281:18723-18733. [Issue cover is CDO structure]
- Dominy, J.E. Jr., Simmons, C.R., Karplus, P.A., Gehring, A.M. and Stipanuk, M.H. 2006. Identification and characterization of bacterial cysteine dioxygenases: a new route of cysteine degradation for eubacteria. J. Bacteriol. 188:5561-5569.
- Stipanuk, M.H., Dominy, J.E. Jr., Lee, J-I. and Coloso, R.M. 2006. Mammalian cysteine metabolism: new insights into regulation of cysteine metabolism. J. Nutr. 136:1652S-1659S.
- Ko, K.S., Torres, C.L., Fascetti, A.J., Stipanuk, M.H., Hirschberger, L. and Rogers, Q.R. 2006. Copper-deficiency does not lead to taurine deficiency in rat. J. Nutr. 583:37-47.
- Stipanuk, M.H. and Watford, M. 2006. Amino acid metabolism. In: Biochemical, Physiological & Molecular Aspects of Human Nutrition (M.H. Stipanuk, ed.), Saunders/Elsevier, St. Louis, pp. 320-418.
- Stipanuk, M.H. 2006. Nutrients: History and definitions. In: Biochemical, Physiological & Molecular Aspects of Human Nutrition (M.H. Stipanuk, ed.), Saunders/Elsevier, St. Louis, pp. 3-12.
- Lewis, B.A. and Stipanuk, M.H. 2006. Structure, nomenclature, and properties of carbohydrates. In: Biochemical, Physiological & Molecular Aspects of Human Nutrition (M.H. Stipanuk, ed.), Saunders/Elsevier, St. Louis, pp. 67-89.
- Stipanuk, M.H. 2006. Protein and amino acid requirements. In: Biochemical, Physiological & Molecular Aspects of Human Nutrition (M.H. Stipanuk, ed.), Saunders/Elsevier, St. Louis, pp. 419-448.
- Stipanuk, M.H. 2006. Regulation of fuel utilization in response to exercise. In: Biochemical, Physiological & Molecular Aspects of Human Nutrition (M.H. Stipanuk, ed.), Saunders/Elsevier, St. Louis, pp. 566-589.
- Stipanuk, M.H. 2006. Disturbances of energy balance. In: Biochemical, Physiological & Molecular Aspects of Human Nutrition (M.H. Stipanuk, ed.), Saunders/Elsevier, St. Louis, pp. 640-660.
- Stipanuk, M.H. 2006. Transamination. In: McGraw Hill Encyclopedia of Science & Technology, 10th edition, McGraw Hill, pp. 540-543.
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Progress 01/01/05 to 12/31/05
Outputs
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 regulation 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 autoxidation of cysteine to form cysteine and reactive oxygen species, 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. Glutathione synthesis increases when intracellular cysteine levels increase, due to increased saturation of glutamate-cysteine ligase
(GCL) with cysteine, and this contributes to removal of excess cysteine. When cysteine levels drop, GCL activity increases and the increased capacity for glutathione synthesis facilitates conservation of cysteine in the form of glutathione (although the absolute rate of glutathione synthesis still decreases due to the lack of substrate). This increase in GCL activity is dependent upon upregulation of expression of both the catalytic and modifier subunits of GCL, resulting in an increase in total catalytic subunit plus an increase in the catalytic efficiency of the enzyme. Cysteine catabolism is tightly regulated via regulation of cysteine dioxygenase levels in the liver, with the turnover of cysteinesulfinate 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 cysteine dioxygenase ubiquitination and, hence, degradation. Because the response of hepatic
cysteine dioxygenase to an increase in cysteine or methionine load is rapid (i.e., less than 24 h to reach new steady-state in the rat) and large (i.e., greater than 30-fold increase in cysteine dioxygenase protein in the rat), 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 cysteine dioxygenase, will be at greater risk of excess cysteine intake. Furthermore, it is possible that tissue or plasma levels of hypotaurine, a metabolite of the cysteine-cysteinesulfinate-hypotaurine-taurine pathway, 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 cysteine dioxygenase protein level and to the tissue concentration of cysteine.
Impacts
Cysteine dioxygenase plays a crucial role in regulating cellular thiol levels. In is now known that thiol redox state is involved in regulation of the function of a number of cellular proteins including transcription factors. Recent evidence indicates that production of H2S from cysteine has important physiological roles in nervous tissue and smooth muscle. Regulation of cysteine levels is largely accomplished by regulation of hepatic cysteine dioxygenase activity. Cysteine dioxygenase activity is regulated in a cysteine-responsive manner via the ubiquitin-proteasome pathway of protein degradation. This is the first example we know of where an enzyme involved in intermediary metabolism has been shown to be upregulated by its substrate via a block in ubiquitylation of the enzyme. Understanding the mechanism of the regulation of cysteine dioxygenase has the potential to improve the nutrition of both individuals in both health and disease.
Publications
- Simmons, C.R., Hao, Q. and Stipanuk, M.H. 2005. Crystallization and preliminary x-ray analysis of purified recombinant cysteine dioxygenase, a non-heme metalloenzyme necessary for regulation of cellular cysteine levels. Acta Crystallog. F61:1013-1016.
- Lee, J-I., Kang, J., and Stipanuk, M.H. 2005. Differential regulation of glutamate-cysteine ligase subunit expression and increased holoenzyme formation in response to cysteine deprivation. Biochem. J. 393:181-190.
- Dominy, J.E., Hirschberger, L.L., Coloso, R. M., and Stipanuk, M.H. 2006. Regulation of cysteine dioxygenase degradation is mediated by intracellular cysteine levels and the ubiquitin-26S proteasome system in the living rat. Biochem. J. 394:267-273.
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Progress 01/01/04 to 12/31/04
Outputs
Cysteine dioxygenase (CDO) catalyzes the first step of cysteine catabolism, its oxidation to cysteinesulfinate. This iron metalloenzyme plays a key role in cysteine catablism, in provision of cysteine carbon for gluconeogenesis or oxidative metabolism, in taurine synthesis, and in supply of inorganic sulfur for sulfation reactions. The regulation of CDO in response to diet is robust, with CDO activity barely detectable in liver of animals fed low protein diets and increasing up to 170-fold in rats fed diets containing high levels of protein or sulfur amino acids. It seems likely that the robust response of hepatic CDO to an increase in cysteine supply may play an essential role in the protection of other tissues from the potentially toxic effects of cysteine. On the other hand, CDO competes with protein synthesis and glutathione synthesis as major consumers of cysteine, and low CDO activity would conserve cysteine for these uses. The rate of CDO degradation is
decreased in the presence of elevated cysteine levels in primary rat hepatocytes, HepG2 (human hepatoma) cells expressing recombinant CDO, and in intact rats. CDO degradation is effectively blocked by proteasome inhibitors (proteasome inhibitor 1 and/or lactacystin) in each of these systems. Cysteamine and homocysteine, unlike most other thiols, reducing agents, and cysteine analogs, also blocked the polyubiquitination and degradation of CDO. In order to further study the effect of cysteamine, we needed to develop an HPLC method for measurement of cysteamine. We have accomplished this and now use iodoacetate to block the sulfhydryl group of cysteamine, derivatize cysteamine with o-phthalaldehyde, and achieve good separation and quantification of cysteamine on a C18 column. In studies with rat hepatocytes or transfected HepG2 cells, cysteamine, at equimolar concentrations in the medium, is as effective as cysteine in blocking CDO degradation. There is a dose-response effect with both
cysteine and cysteamine that is very similar to that for cysteine: the maximal response occurs between 0.1 and 0.5 mmol/L. Interestingly, the cysteine concentration in the portal blood of intact rats fed a high cysteine diet was 0.2 mmol/L compared to 0.1 mmol/L in portal blood of rats fed a basal low protein diet or in the arterial blood of rats fed with diet. When fed in the diet at 8 g cysteine per kg diet (or an equimolar amount of cysteamine) on top of a 10 percent casein diet, both cysteine and cysteamine resulted in intracellular concentrations of about 0.05 mmol/kg liver cells. However, plasma concentrations of cysteine were 4- to 5-times those of cysteamine (0.2 mM vs. 0.04 mM for cysteamine), and cysteine was much more effective than cysteamine at blocking CDO degradation in vivo. These observations suggest than the mechanism of regulation of CDO polyubiquitination and degradation may not be directly due to the cysteine molecule. Studies of the structure of CDO, development
of CDO transgenic mice, and studies of the role of cysteamine in taurine production are also underway.
Impacts
Cysteine dioxygenase plays a crucial role in regulating cellular thiol levels. In is now known that thiol redox state is involved in regulation of the function of a number of cellular proteins including transcription factors. Recent evidence indicates that production of H2S from cysteine has important physiological roles in nervous tissue and smooth muscle. Regulation of cysteine levels is largely accomplished by regulation of hepatic cysteine dioxygenase activity. Cysteine dioxygenase activity is regulated in a cysteine-responsive manner via the ubiquitin-proteasome pathway of protein degradation. This is the first example we know of where an enzyme involved in intermediary metabolism has been shown to be upregulated by its substrate via a block in ubiquitylation of the enzyme. Understanding the mechanism of the regulation of cysteine dioxygenase has the potential to improve the nutrition of both individuals in both health and disease.
Publications
- Stipanuk, M.H. 2004. Sulfur Amino Acid Metabolism: Pathways for Production and Removal of Homocysteine and Cystine. Annu. Rev. Nutr. 24:539-577.
- Dominy, J. E., Stipanuk, M. H. 2004. New roles for cysteine and transsulfuration enzymes: production of H2S, a neuromodulator and smooth muscle relaxant. Nutr. Rev. 62:348-353.
- Lee, J.-I., Londono, M. P., Hirschberger, L. L., and Stipanuk, M. H. 2004. Regulation of cysteine dioxygenase and gamma-glutamylcysteine synthetase is associated with hepatic cysteine level. J Nutr Biochem 15: 112-122.
- Stipanuk, M. H., Hirschberger, L. L., Londono, M. P., Cresenzi, C. L., and Yu, A. F. 2004. The ubiquitin-proteasome system is responsible for cysteine-responsive regulation of cysteine dioxygenase concentration in liver. Am J Physiol Endocrinol Metab 286: E439-E448.
- Stipanuk, M.H. 2004. Homocysteine, Cysteine and Taurine. Modern Nutrition in Health and Disease, 10th edition, in press.
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Progress 01/01/03 to 12/31/03
Outputs
Cysteine dioxygenase catalyzes the first step in the major catabolic pathway for the amino acid cysteine. Cysteine itself and H2S produced from cysteine by constitutive, low-capacity pathways are cytotoxic and neuroexcitotoxic. Thus, robust regulation of cysteine dioxygenase is necessary to control tissue cysteine concentrations within narrow limits. A marked increase in cysteine dioxygenase level occurred when rats were switched from a low protein diet to a high protein diet, and the cysteine dioxygenase level dropped rapidly when rats fed a high protein diet were switched to a low protein diet. Hepatic cysteine levels reflected the protein/sulfur amino acid intake but remained below 0.1 mmol/g. Cysteine dioxygenase mRNA levels did not change with the diet switches. Studies with hepatocytes and intact rats have indicated that cysteine, not methionine or a methionine or cysteine metabolite, mediates the regulation of hepatic cysteine dioxygenase levels. In studies
with rat hepatocytes in culture, cysteamine was the only cysteine analog not capable of being converted to cysteine that was effective in bringing about an accumulation of cysteine dioxygenase. Further studies have indicated that cysteine dioxygenase is degraded by the ubiquitin-proteasome system. In the presence of cysteine or cysteamine, ubiquitylation of cysteine dioxygenase is inhibited and, thus, cysteine dioxygenase is not targeted to the proteasome for degradation. In the absence of cysteine, ubiquitylation of cysteine dioxygenase occurs and the cysteine dioxygenase-polyubiquitin conjugate is recognized by the 26S proteasome and cysteine dioxygenase is degraded. This is the first example we know of where an enzyme involved in intermediary metabolism has been shown to be upregulated by its substrate via a block in ubiquitylation of the enzyme.
Impacts
Cysteine dioxygenase plays a crucial role in regulating cellular thiol levels. In is now known that thiol redox state is involved in regulation of the function of a number of cellular proteins including transcription factors. Recent evidence indicates that production of H2S from cysteine has important physiological roles in nervous tissue and smooth muscle. Regulation of cysteine levels is largely accomplished by regulation of hepatic cysteine dioxygenase activity. Cysteine dioxygenase activity is regulated in a cysteine-responsive manner via the ubiquitin-proteasome pathway of protein degradation. This is the first example we know of where an enzyme involved in intermediary metabolism has been shown to be upregulated by its substrate via a block in ubiquitylation of the enzyme. Understanding the mechanism of the regulation of cysteine dioxygenase has the potential to improve the nutrition of both individuals in both health and disease.
Publications
- Cresenzi, C. L., Lee, J.-I., and Stipanuk, M. H. 2003. Cysteine is the metabolic signal responsible for dietary regulation of cysteine dioxygenase and glutamate cysteine ligase in vivo. J Nutr 133:2697-2702.
- Stipanuk, M. H. 2003. Role of the liver in regulation of body cysteine and taurine levels. Neurochem Res: 29: 105-110.
- Lee, J.-I., Londono, M. P., Hirschberger, L. L., and Stipanuk, M. H. 2004. Regulation of cysteine dioxygenase and g-glutamylcysteine synthetase is associated with hepatic cysteine level. J Nutr Biochem: in press.
- Stipanuk, M. H., Hirschberger, L. L., Londono, M. P., Cresenzi, C. L., and Yu, A. F. 2004. The ubiquitin-proteasome system is responsible for cysteine-responsive regulation of cysteine dioxygenase concentration in liver. Am J Physiol: in press.
- Lee, J.I., Londono, M., and Stipanuk, M.H. 2003. Time-course of changes in g-glutamylcysteine synthetase and cysteine dioxygenase. FASEB J.17: 431.6.
- Stipanuk, M.H., Londono, M., Hirschberger, L.L. 2003. Cysteine-responsive regulation of hepatic cysteine dioxygenase by the ubiquitin-proteasome system. FASEB J.17: 449.4.
- Stipanuk, M.H., Wang, L., Hickey, C. and Hirschberger, L.L. 2003. Molecular mass of active mammalian cysteine dioxygenase. FASEB J. 17: 449.3.
- Ko, K. S., Torres, C., L., Fascetti, A. J., Stipanuk, M. H., Rogers, Q. R. 2004. Plasma taurine does not decrease in copper-deficient rats. FASEB J., in press
- Stipanuk, M. H. Londono, M., Hirschberger, L. L., Wang, L., and Hickey, C. 2004. Evidence for expression of a single distinct form of mammalian cysteine dioxygenase. Amino Acids: in press.
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Progress 01/01/02 to 12/31/02
Outputs
Cysteine dioxygenase mRNA is expressed in liver, kidney, lung, and brain, and the abundance of cysteine dioxygenase mRNA in these tissues is proportional to the amount of cysteine dioxygenase activity in these tissues. In liver, however, while cysteine dioxygenase mRNA level remained constant, cysteine dioxygenase activity and concentration decreased when dietary sulfur amino acids or protein were decreased to below the requirement or increased when dietary sulfur amino acids or protein were increased to above the requirement level. When rats were switched from a low protein diet to a high protein diet, hepatic cysteine dioxygenase increased markedly and had nearly reached the plateau value by 24 h. Similarly, when rats were switched from a high protein diet to a low protein diet, hepatic cysteine dioxygenase decreased rapidly and was very low by 12 h after the diet switch. Cysteine dioxygenase mRNA levels did not change with the diet switches. In studies with rat
hepatocytes in culture, cysteine and cysteamine (decarboxylated cysteine) were equally effective in bringing about the accumulation of cysteine dioxygenase; compounds with blocked or oxidized sulfhydryl groups or thiol reagents including 3-mercaptoethanol (cysteamine without the amino group) were not effective. Addition of inhibitors or the 26S proteasome, but not of lysosomal proteases or calcium-dependent calpains, resulted in increases in cysteine dioxygenase abundance, suggesting that cysteine degradation occurs via the ubiquitin-proteasome system. Further studies have indicated that indeed, cysteine dioxygenase is degraded by the ubiquitin-proteasome system. In the presence of cysteine or cysteamine, ubiquitylation of cysteine dioxygenase is inhibited and, thus, cysteine dioxygenase is not targeted to the proteasome for degradation. In the absence of cysteine, ubiquitylation of cysteine dioxygenase occurs and the cysteine dioxygenase-polyubiquitin conjugate is recognized by the
26S proteasome and cysteine dioxygenase is degraded. This is the first example we know of where an enzyme involved in intermediary metabolism has been shown to be upregulated by its substrate via a block in ubiquitylation of the enzyme.
Impacts
Cysteine dioxygenase plays a crucial role in regulating cellular thiol levels. Careful regulationof body cysteine concentrations is essential because cysteine is needed for synthesis of protein, glutathione, taurine, coenzyme A, and inorganic sulfur, but excess levels of cysteine are neuroexcitotoxic. The incidence of alterations in cysteine catabolism by cysteine dioxygenase is markedly elevated in populations with rheumatoid arthritis, Parkinson's disease, and motor neurone disease. Understanding the role and regulation of cysteine dioxygenase will enable improvement of nutrition in both healthy and diseased populations.
Publications
- Kwon, Y.H. and Stipanuk, M.H. 2001. Cysteine regulates expression of cysteine dioxygenase and g-glutamylcysteine synthetase in cultured rat hepatocytes. Am J Physiol Endocrinol Metab 280(5): E804-E815.
- Hirschberger L.L., Daval, S., Stover, P.J. and Stipanuk, M.H. 2001. Murine cysteine dioxygenase gene: structural organization, tissue-specific expression and promoter identification. Gene 277(1-2): 153-161.
- Bella D.L., Hirschberger, L.L., Kwon Y.H. and Stipanuk, M.H. 2002. Cysteine metabolism in periportal and perivenous hepatocytes: perivenous cells have greater capacity for glutathione production and taurine synthesis but not for cysteine catabolism. Amino Acids 23: 453-458.
- Stipanuk M.H., Londono, M., Lee, J-I., Hu, M. and Yu, A.F. 2002. Enzymes and metabolites of cysteine metabolism in nonhepatic tissues of rats show little response to changes in dietary protein or sulfur amino acid levels. J. Nutr. 132: 3369-3378.
- Stipanuk, M.H., Londono, M., Hirschberger, L.L., Hickey, C., Thiel, D.J. and Wang, L. 2003. Evidence for expression of a single distinct form of mammalian cysteine dioxygenase. Amino Acids: in press.
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Progress 01/01/01 to 12/31/01
Outputs
Cysteine dioxygenase mRNA is expressed in liver, kidney, lung, and brain, and the abundance of cysteine dioxygenase mRNA in these tissues is proportional to the amount cysteine dioxygenase cysteine dioxygenase activity in these tissues. In liver, however, while cysteine dioxygenase mRNA level remained constant, cysteine dioxygenase activity and concentration decreased when dietary sulfur amino acids or protein were decreased to below the requirement or increased when dietary sulfur amino acids or protein were increased to above the requirement level. When rats were switched from a low protein diet to a high protein diet, hepatic cysteine dioxygenase increased markedly and had nearly reached the plateau value by 24 h. Similarly, when rats were switched from a high protein diet to a low protein diet, hepatic cysteine dioxygenase decreased rapidly and was very low by 12 h after the diet switch. Cysteine dioxygenase mRNA levels did not change with the diet switches. In
studies with rat hepatocytes in culture, cysteine or cysteine analogs that contained a free sulfhydryl group caused accumulation of cysteine dioxygenase. Addition of proteasome inhibitors also resulted in increases in cysteine dioxygenase abundance. Our results suggest that regulation of cysteine dioxygenase levels may involve regulation of its degradation and perhaps involves stabilization of the enzyme by its substrate. We are developing cysteine dioxygenase expression and over-expression systems with which we will investigate the role of protein turnover and the ubiquitin-proteasome system in regulation of cysteine dioxygenase levels.
Impacts
Understanding the role of cysteine dioxygenase in regulation of tissue cysteine concentrations has health implications. It is essential to protect the body from high cysteine levels, which are neurotoxic, and, at the same time, it is necessary to have a mechanism to conserve cysteine for synthesis of glutathione and other essential metabolites. Cysteine dioxygenase undergoes a rapid and marked regulatory response when cysteine intake changes. Heterogeneity in the expression and/or regulation of cysteine dioxygenase may be a component of the etiology of certain chronic degenerative diseases such as Parkinson's disease and rheumatoid arthritis.
Publications
- Kwon, Y. H., and Stipanuk, M. H. 2001. Cysteine regulates expression of and gamma-glutamylcysteine synthetase in cultured rat hepatocytes. Am. J. Physiol., 280: E804-E815.
- Hirschberger, L. L., Daval, S., Stover, P. J., and Stipanuk, M. H. 2001. Murine cysteine dioxygenase gene: structure organization, tissue-specific expression and promoter identification. Gene 277: 153-161.
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Progress 01/01/00 to 12/31/00
Outputs
Cysteine dioxygenase mRNA is expressed in liver, kidney, and lung, but these tissues contain low levels of active enzyme when cysteine levels are low. Cysteine dioxygenase accumulates as dietary sulfur amino acid level increases. In rats fed diets that contained various levels of sulfur amino acids, hepatic cysteine concentration increased from 0.02 mmol/kg in liver of rats fed the low protein (10% casein) diet to 0.08 mmol/kg in liver of rats fed a high protein (40% casein) or high sulfur amino acid diet (10% casein + 8 g L-cystine or 10 g L-methionine); cysteine dioxygenase activity and protein increased by more than 30-fold. In studies with rat hepatocytes in primary culture, cysteine dioxygenase mRNA, protein, and activity were lost with time in culture when standard medium was used, but cysteine dioxygenase accumulated in cells cultured with excess sulfur amino acids. Our results suggest that regulation of cysteine dioxygenase activity occurs posttranslationally
and likely involves stabilization of cysteine dioxygenase by cysteine. Tissue specific regulation of cysteine dioxygenase gene expression also clearly occurs because only a few tissues have detectable levels of cysteine dioxygenase mRNA. Tissue mRNA concentration does not respond to variations in dietary sulfur amino acid intake in vivo although slight increases occur in hepatocytes cultured in medium with excess sulfur amino acids. We have cloned and sequenced the mouse cysteine dioxygenase gene, which is homologous to the rat and human gene, and we are presently making a construct to use in generating a cysteine dioxygenase transgenic/knockout mouse model for further studies of the function and health-significance of cysteine dioxygenase.
Impacts
Cysteine dioxygenase undergoes a strong upregulation in the presence of excess cysteine. This regulation appears to occur at the level of protein turnover with cysteine stabilizing the protein. Cysteine is both neurotoxic and cytotoxic, so rapid removal of excess is critical. On the other hand, cysteine is essential for protein and glutathione synthesis and for formation of taurine and inorganic sulfate and must be conserved when the supply is low. Because cysteine dioxygenase polymorphisms have been suggested to be associated with the incidence or progression of certain neurological diseases, it is important to develop a model (the mouse cysteine dioxygenase transgenic/knockout model)that will allow us to study this association.
Publications
- Kwon, Y. H., and Stipanuk, M. H. 2001 Cysteine regulates expression of cysteine dioxygenase and gamma-glutamylcysteine synthetase in cultured rat hepatocytes. Am. J. Physiol., in press.
- Ohta, J., Kwon, Y. H., and Stipanuk, M. H. 2000 Cysteine dioxygenase and gamma-glutamylcysteine synthetase activities in primary cultured hepatocytes respond to sulfur amino acid supplementation in a reciprocal manner. Amino Acids 19: 705-728.
- Stipanuk, M.H., Hirschberger, L.L., Londono, M., Hu, M., and Lee, Y-I. 2001 Tissue-specific responses to increases in dietary protein or sulfur amino acids. FASEB J., in press.
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