Metabolism of Sulfur-Containing Amino Acids: How the Body Copes with Excess Methionine, Cysteine, and Sulfide.

Metabolism of excess methionine (Met) to homocysteine (Hcy) by transmethylation is facilitated by the expression of methionine adenosyltransferase (MAT) I/III and glycine N-methyltransferase (GNMT) in liver, and a lack of either enzyme results in hypermethioninemia despite normal concentrations of MATII and methyltransferases other than GNMT. The further metabolism of Hcy by the transsulfuration pathway is facilitated by activation of cystathionine β-synthase (CBS) by S-adenosylmethionine (SAM) as well as the relatively high KM of CBS for Hcy. Transmethylation plus transsulfuration effects catabolism of the Met molecule along with transfer of the sulfur atom of Met to serine to synthesize cysteine (Cys).

Spectroscopic Investigation of Cysteamine Dioxygenase.

Thiol dioxygenases are mononuclear non-heme Fe-dependent metalloenzymes that initiate the oxidative catabolism of thiol-containing substrates to their respective sulfinates. Cysteine dioxygenase (CDO), the best characterized mammalian thiol dioxygenase, contains a three-histidine (3-His) coordination environment rather than the 2-His-1-carboxylate facial triad seen in most mononuclear non-heme Fe enzymes. A similar 3-His active site is found in the bacterial thiol dioxygenase 3-mercaptopropionate dioxygenase (MDO), which converts 3-mercaptopropionate into 3-sulfinopropionic acid as part of the bacterial sulfur metabolism pathway.

Effects of single amino acid deficiency on mRNA translation are markedly different for methionine versus leucine.

Although amino acids are known regulators of translation, the unique contributions of specific amino acids are not well understood. We compared effects of culturing HEK293T cells in medium lacking either leucine, methionine, histidine, or arginine on eIF2 and 4EBP1 phosphorylation and measures of mRNA translation. Methionine starvation caused the most drastic decrease in translation as assessed by polysome formation, ribosome profiling, and a measure of protein synthesis (puromycin-labeled polypeptides) but had no significant effect on eIF2 phosphorylation, 4EBP1 hyperphosphorylation or 4EBP1 binding to eIF4E.

Cysteine dioxygenase is essential for mouse sperm osmoadaptation and male fertility.

Sperm entering the epididymis are immotile and cannot respond to stimuli that will enable them to fertilize. The epididymis is a highly complex organ, with multiple histological zones and cell types that together change the composition and functional abilities of sperm through poorly understood mechanisms. Sperm take up taurine during epididymal transit, which may play antioxidant or osmoregulatory roles.

Identification of Taurine-Responsive Genes in Murine Liver Using the Cdo1-Null Mouse Model.

The cysteine dioxygenase (Cdo1)-null mouse is unable to synthesize hypotaurine and taurine by the cysteine/cysteine sulfinate pathway and has very low taurine levels in all tissues. The lack of taurine is associated with a lack of taurine conjugation of bile acids, a dramatic increase in the total and unconjugated hepatic bile acid pools, and an increase in betaine and other molecules that serve as organic osmolytes. We used the Cdo1-mouse model to determine the effects of taurine deficiency on expression of proteins involved in sulfur amino acid and bile acid metabolism.

GCN2- and eIF2α-phosphorylation-independent, but ATF4-dependent, induction of CARE-containing genes in methionine-deficient cells.

Amino-acid deprivation is sensed by the eIF2α kinase GCN2. Under conditions of essential amino-acid limitation, GCN2 phosphorylates eIF2α, inhibiting the formation of a new ternary complex and hence mRNA translation initiation. While decreasing global mRNA translation, eIF2α phosphorylation also increases the translation of the integrated stress response (ISR) transcription factor ATF4, which increases the expression of many stress response genes that contain a C/EBP-ATF response element (CARE), including Atf4, 4Ebp1, Asns, and Chop.

Structure-Based Insights into the Role of the Cys-Tyr Crosslink and Inhibitor Recognition by Mammalian Cysteine Dioxygenase.

In mammals, the non-heme iron enzyme cysteine dioxygenase (CDO) helps regulate Cys levels through converting Cys to Cys sulfinic acid. Its activity is in part modulated by the formation of a Cys93-Tyr157 crosslink that increases its catalytic efficiency over 10-fold. Here, 21 high-resolution mammalian CDO structures are used to gain insight into how the Cys-Tyr crosslink promotes activity and how select competitive inhibitors bind.

Effects of a block in cysteine catabolism on energy balance and fat metabolism in mice.

To gain further insights into the effects of elevated cysteine levels on energy metabolism and the possible mechanisms underlying these effects, we conducted studies in cysteine dioxygenase (Cdo1)-null mice. Cysteine dioxygenase (CDO) catalyzes the first step of the major pathway for cysteine catabolism. When CDO is absent, tissue and plasma cysteine levels are elevated, resulting in enhanced flux of cysteine through desulfhydration reactions.

Amino acids in healthy aging skeletal muscle.

Life expectancy in the U.S. and globally continues to increase.

Downregulation of hepatic betaine:homocysteine methyltransferase (BHMT) expression in taurine-deficient mice is reversed by taurine supplementation in vivo.

The cysteine dioxygenase (Cdo1)-null and the cysteine sulfinic acid decarboxylase (Csad)-null mouse are not able to synthesize hypotaurine/taurine by the cysteine/cysteine sulfinate pathway and have very low tissue taurine levels. These mice provide excellent models for studying the effects of taurine on biological processes. Using these mouse models, we identified betaine:homocysteine methyltransferase (BHMT) as a protein whose in vivo expression is robustly regulated by taurine.

Propargylglycine inhibits hypotaurine/taurine synthesis and elevates cystathionine and homocysteine concentrations in primary mouse hepatocytes.

Our investigation showed that hepatocytes isolated from cysteine dioxygenase knockout mice (Cdo1(-/-)) had lower levels of hypotaurine and taurine than Cdo1 (+/+) hepatocytes. Interestingly, hypotaurine accumulates in cultured wild-type hepatocytes. DL-propargylglycine (PPG, inhibitor of cystathionine γ-lyase and H2S production) dramatically decreased both taurine and hypotaurine levels in wild-type hepatocytes compared to untreated cells.

The first international mini-symposium on methionine restriction and lifespan.

It has been 20 years since the Orentreich Foundation for the Advancement of Science, under the leadership Dr. Norman Orentreich, first reported that low methionine (Met) ingestion by rats extends lifespan (Orentreich et al., 1993).

Primary hepatocytes from mice lacking cysteine dioxygenase show increased cysteine concentrations and higher rates of metabolism of cysteine to hydrogen sulfide and thiosulfate.

The oxidation of cysteine in mammalian cells occurs by two routes: a highly regulated direct oxidation pathway in which the first step is catalyzed by cysteine dioxygenase (CDO) and by desulfhydration-oxidation pathways in which the sulfur is released in a reduced oxidation state. To assess the effect of a lack of CDO on production of hydrogen sulfide (H2S) and thiosulfate (an intermediate in the oxidation of H2S to sulfate) and to explore the roles of both cystathionine γ-lyase (CTH) and cystathionine β-synthase (CBS) in cysteine desulfhydration by liver, we investigated the metabolism of cysteine in hepatocytes isolated from Cdo1-null and wild-type mice. Hepatocytes from Cdo1-null mice produced more H2S and thiosulfate than did hepatocytes from wild-type mice.

Upregulation of capacity for glutathione synthesis in response to amino acid deprivation: regulation of glutamate-cysteine ligase subunits.

Using HepG2/C3A cells and MEFs, we investigated whether induction of GSH synthesis in response to sulfur amino acid deficiency is mediated by the decrease in cysteine levels or whether it requires a decrease in GSH levels per se. Both the glutamate-cysteine ligase catalytic (GCLC) and modifier (GCLM) subunit mRNA levels were upregulated in response to a lack of cysteine or other essential amino acids, independent of GSH levels. This upregulation did not occur in MEFs lacking GCN2 (general control non-derepressible 2, also known as eIF2α kinase 4) or in cells expressing mutant eIF2α lacking the eIF2α kinase Ser(51) phosphorylation site, indicating that expression of both GCLC and GCLM was mediated by the GCN2/ATF4 stress response pathway.

Total 4EBP1 Is Elevated in Liver of Rats in Response to Low Sulfur Amino Acid Intake.

Translation initiation is known to be regulated by the binding of eukaryotic initiation factor 4E (eIF4E) by binding proteins (4EBPs), and there is evidence that amino acid deprivation and other cellular stresses upregulate 4EBP1 expression. To pursue the question of whether diets limited in an essential amino acid lead to induction of 4EBP1 expression in vivo, diets that varied in methionine and cystine content were fed to rats for 7 days, and 4EBP1 mRNA and protein levels and 4EBP1 phosphorylation state were determined. Total 4EBP1 mRNA and protein abundance increased in liver of rats with severely deficient intakes of sulfur amino acids (0.

Cysteine sulfinic acid decarboxylase regulation: A role for farnesoid X receptor and small heterodimer partner in murine hepatic taurine metabolism.

Aim: Bile acid synthesis is regulated by nuclear receptors including farnesoid X receptor (FXR) and small heterodimer partner (SHP), and by fibroblast growth factor 15/19 (FGF15/19). We hypothesized that hepatic cysteine sulfinic acid decarboxylase (CSAD) (a key enzyme in taurine synthesis) is regulated by bile acids (BA). The aim of this study was to investigate CSAD regulation by BA dependent regulatory mechanisms.

Cysteine dioxygenase structures from pH4 to 9: consistent cys-persulfenate formation at intermediate pH and a Cys-bound enzyme at higher pH.

Mammalian cysteine dioxygenase (CDO) is a mononuclear non-heme iron protein that catalyzes the conversion of cysteine (Cys) to cysteine sulfinic acid by an unclarified mechanism. One structural study revealed that a Cys-persulfenate (or Cys-persulfenic acid) formed in the active site, but quantum mechanical calculations have been used to support arguments that it is not an energetically feasible reaction intermediate. Here, we report a series of high-resolution structures of CDO soaked with Cys at pH values from 4 to 9.

The cysteine dioxgenase knockout mouse: altered cysteine metabolism in nonhepatic tissues leads to excess H2S/HS(-) production and evidence of pancreatic and lung toxicity.

Aims: To define the consequences of loss of cysteine dioxygenase (CDO) on cysteine metabolism at the tissue level, we determined levels of relevant metabolites and enzymes and evidence of H2S/HS(-) (gaseous hydrogen sulfide and its conjugate base) toxicity in liver, pancreas, kidney, and lung of CDO(-/-) mice that were fed either a taurine-free or taurine-supplemented diet.

Results: CDO(-/-) mice had low tissue and serum taurine and hypotaurine levels and high tissue levels of cysteine, consistent with the loss of CDO. CDO(-/-) mice had elevated urinary excretion of thiosulfate, high tissue and serum cystathionine and lanthionine levels, and evidence of inhibition and destabilization of cytochrome c oxidase, which is consistent with excess production of H2S/HS(-).

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).

Protein intake and muscle strength in older persons: does inflammation matter?

Objectives: To examine whether protein intake is associated with change in muscle strength in older persons. Because systemic inflammation has been associated with protein catabolism, the study also evaluated whether a synergistic effect exists between protein intake and inflammatory markers on change in muscle strength.

Design: Longitudinal.

Knockout of the murine cysteine dioxygenase gene results in severe impairment in ability to synthesize taurine and an increased catabolism of cysteine to hydrogen sulfide.

Cysteine homeostasis is dependent on the regulation of cysteine dioxygenase (CDO) in response to changes in sulfur amino acid intake. CDO oxidizes cysteine to cysteinesulfinate, which is further metabolized to either taurine or to pyruvate plus sulfate. To gain insight into the physiological function of CDO and the consequence of a loss of CDO activity, mice carrying a null CDO allele (CDO(+/-) mice) were crossed to generate CDO(-/-), CDO(+/-), and CDO(+/+) mice.

Gene expression and integrated stress response in HepG2/C3A cells cultured in amino acid deficient medium.

The integrated stress response (ISR), a defense mechanism cells employ when under stress (e.g., amino acid deprivation), causes suppression of global protein synthesis along with the paradoxical increased expression of a host of proteins that are useful in combating various stresses.

Growing rats respond to a sulfur amino acid-deficient diet by phosphorylation of the alpha subunit of eukaryotic initiation factor 2 heterotrimeric complex and induction of adaptive components of the integrated stress response.

Mammalian cells respond to various kinds of stress, including nutritional stress, via pathways that are initiated by phosphorylation of the alpha subunit of the eukaryotic initiation factor 2 complex (eIF2alpha). Because the models used to study eIF2alpha-kinase-mediated responses to amino acid deficiency have commonly used media or diets devoid of 1 or more essential amino acids, we asked whether eIF2alpha-kinase-mediated responses would be induced in animals fed a more typical diet that was not as imbalanced as one in which 1 essential amino acid is totally absent. To answer this question, we fed rats soy protein-based diets that were either adequate or limiting in sulfur-containing amino acids (SAA).