Relicardo M. Coloso

Mammalian Cysteine Metabolism: New Insights into Regulation of Cysteine Metabolism

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. Cysteine catabolism is tightly regulated via regulation of cysteine dioxygenase (CDO) levels in the liver, with the turnover of CDO protein 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 degradation. Glutathione synthesis also increases when intracellular cysteine levels increase as a result of 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 because of the lack of substrate). This increase in GCL activity is dependent on up-regulation 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. An important role of cysteine utilization for coenzyme A synthesis in maintaining cellular cysteine levels in some tissues, and a possible connection between the necessity of controlling cellular cysteine levels to regulate the rate of hydrogen sulfide production, have been suggested by recent literature and are areas that deserve further study.

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

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

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

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

Metabolism of Cysteine to Taurine by Rat Hepatocytes

During the past two decades, many investigators have assumed that the major locus of regulation of cysteine catabolism is the partitioning of cysteinesulfinate between its decarboxylation and transamination pathways. Hepatic cysteinesulfinate decarboxylase activity correlates well with the capacity of animals to synthesize taurine1–4, and low cysteinesulfinate decarboxylase activity in the cat has been associated with its nutritional requirement for dietary taurine5. More recent studies in our laboratory have indicated that cysteinesulfinate-independent pathways also play a major role in cysteine metabolism6,7. In contrast to cysteinesulfinate-dependent metabolism of cysteine, which leads to both taurine and sulfate production, the cysteinesulfinate-independent pathways all result in release of reduced inorganic sulfur and its subsequent oxidation to sulfate. This evidence revealing a contribution of cysteinesulfinate-independent pathways to cysteine catabolism suggested that partitioning of cysteine between cysteinesulfinate formation and metabolism by cysteinesulfinate-independent pathways may also be important in the regulation of cysteine metabolism to taurine.

The effect of dietary protein-energy levels on growth and metabolism of milkfish (Chanos chanos Forsskal)

Abstract 1. 1. Groups of milkfish juveniles (mean weight, 2.8 g) were fed diets containing white fishmeal and gelatin with varying protein-energy to total metabolizable energy (PE:TME) ratios. 2. 2. Amino acids were incorporated in the diets to stimulate the pattern of milkfish protein. The control diet contained fishmeal as sole protein source and was not supplemented with amino acids. 3. 3. Among the amino acid supplemented diets, best growth was observed at PE:TME ratio of 44.4%. However, the control diet gave better growth rate than any of the amino acid supplemented diets. 4. 4. Specific activities of pyruvate kinase (PK) and glutamate dehydrogenase (GDH) increased significantly with increase in dietary protein-energy level.