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Mass spectrometric analysis of L-cysteine metabolism: physiological role and fate of L-cysteine in the enteric protozoan parasite Entamoeba histolytica.

Jeelani G, Sato D, Soga T, Watanabe H, Nozaki T - MBio (2014)

Bottom Line: Furthermore, T4C and MT4C significantly enhanced trophozoite growth and reduced intracellular reactive oxygen species (ROS) levels when it was added to cultures, suggesting that 2-(R)-thiazolidine-4-carboxylic acids are involved in the defense against oxidative stress.We found that L-cysteine inside the cell rapidly reacts with aldehydes to form 2-(R)-thiazolidine-4-carboxylic acid.We showed that these 2-(R)-thiazolidine-4-carboxylic derivatives serve as an L-cysteine source, promote growth, and protect cells against oxidative stress by scavenging aldehydes and reducing the ROS level.

View Article: PubMed Central - PubMed

Affiliation: Department of Parasitology, National Institute of Infectious Diseases, Shinjuku, Tokyo, Japan.

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Related in: MedlinePlus

Influence of T4C, MT4C, and l-cysteine on the intracellular ROS levels. Trophozoites were cultivated in l-cysteine-deprived BI-S-33 medium for 72 h, and after that, the medium was replaced with l-cysteine-deprived BI-S-33 media containing 2 mM T4C, MT4C, or l-cysteine. After 3 h, approximately 4.0 × 105 cells were then incubated with the dye 2′,7′-DCF-DA for 20 min. The intracellular ROS levels were quantified by determination of DCF fluorescence. Results were normalized with cell numbers and are presented relative to levels in untreated control cells. The means ± SD from three independent experiments performed in triplicate are shown. Statistical comparisons were made by Student’s t test (**, P < 0.01; ***, P < 0.001).
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fig6: Influence of T4C, MT4C, and l-cysteine on the intracellular ROS levels. Trophozoites were cultivated in l-cysteine-deprived BI-S-33 medium for 72 h, and after that, the medium was replaced with l-cysteine-deprived BI-S-33 media containing 2 mM T4C, MT4C, or l-cysteine. After 3 h, approximately 4.0 × 105 cells were then incubated with the dye 2′,7′-DCF-DA for 20 min. The intracellular ROS levels were quantified by determination of DCF fluorescence. Results were normalized with cell numbers and are presented relative to levels in untreated control cells. The means ± SD from three independent experiments performed in triplicate are shown. Statistical comparisons were made by Student’s t test (**, P < 0.01; ***, P < 0.001).

Mentions: 2-(R)-Thiazolidine-4-carboxylic acids, including T4C are cyclic-sulfur-containing amino acids that are analogous in molecular structure to l-proline. It has been shown that T4C can act as an intracellular sulfhydryl antioxidant and a scavenger of free radicals and thereby protect cellular membranes and other oxidation-prone structures in the cell from damage due to oxygen and oxygen-derived free radicals (55). It was shown that T4C stimulates oxygen uptake in rat liver mitochondria (43). As T4C plays an important role in oxidative-defense mechanisms (55), it was of interest to examine the effect of T4C and MT4C on the amount of intracellular ROS. Our previous study showed that when E. histolytica trophozoites were cultured under l-cysteine-limited conditions for 72 h, the intracellular levels of reactive oxygen species increased 4-fold (18). We cultivated trophozoites in l-cysteine-deprived BI-S-33 medium for 72 h, and the medium was replaced with l-cysteine-deprived BI-S-33 medium containing 2 mM T4C, MT4C, or l-cysteine. After 3 h, the relative level of ROS was measured using the fluorescent indicator CM-H 2DCFDA [5-(and-6)-chloromethyl-2′,7′-dichlorodihydrofluorescein diacetate, acetyl ester]. We found that the intracellular levels of reactive oxygen species in trophozoites cultured with 2 mM T4C, MT4C, or l-cysteine were, respectively, approximately 50, 21, or 32% lower than those in control cells (Fig. 6, bar Cys dep). These results suggest that T4C, M4C, and l-cysteine (T4C in particular) are important scavengers of reactive oxygen species in E. histolytica. The level of suppression of ROS by supplemented thiazolidine-4-carboxylic acids in the l-cysteine-deprived culture medium was only partial (<50%). This may be because, besides the thiazolidine-4-carboxylic acids described here, l-cysteine-derived metabolites that are involved in the antioxidant defense mechanism may exist in E. histolytica. Mackenzie and Harris were the first to recognize the therapeutic potential of T4C in animals (43). They noted that T4C is about five times more potent than l-cysteine in preventing massive pleural effusions and death in thiourea-treated rats. It was presumed that T4C, possessing a protected sulfur atom in its ring, opens and frees a sulfhydryl group after entering a liver cell. l-Cysteine, on the other hand, has an unprotected free sulfhydryl group, which is likely to react with oxidants before entering a cell.


Mass spectrometric analysis of L-cysteine metabolism: physiological role and fate of L-cysteine in the enteric protozoan parasite Entamoeba histolytica.

Jeelani G, Sato D, Soga T, Watanabe H, Nozaki T - MBio (2014)

Influence of T4C, MT4C, and l-cysteine on the intracellular ROS levels. Trophozoites were cultivated in l-cysteine-deprived BI-S-33 medium for 72 h, and after that, the medium was replaced with l-cysteine-deprived BI-S-33 media containing 2 mM T4C, MT4C, or l-cysteine. After 3 h, approximately 4.0 × 105 cells were then incubated with the dye 2′,7′-DCF-DA for 20 min. The intracellular ROS levels were quantified by determination of DCF fluorescence. Results were normalized with cell numbers and are presented relative to levels in untreated control cells. The means ± SD from three independent experiments performed in triplicate are shown. Statistical comparisons were made by Student’s t test (**, P < 0.01; ***, P < 0.001).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4222106&req=5

fig6: Influence of T4C, MT4C, and l-cysteine on the intracellular ROS levels. Trophozoites were cultivated in l-cysteine-deprived BI-S-33 medium for 72 h, and after that, the medium was replaced with l-cysteine-deprived BI-S-33 media containing 2 mM T4C, MT4C, or l-cysteine. After 3 h, approximately 4.0 × 105 cells were then incubated with the dye 2′,7′-DCF-DA for 20 min. The intracellular ROS levels were quantified by determination of DCF fluorescence. Results were normalized with cell numbers and are presented relative to levels in untreated control cells. The means ± SD from three independent experiments performed in triplicate are shown. Statistical comparisons were made by Student’s t test (**, P < 0.01; ***, P < 0.001).
Mentions: 2-(R)-Thiazolidine-4-carboxylic acids, including T4C are cyclic-sulfur-containing amino acids that are analogous in molecular structure to l-proline. It has been shown that T4C can act as an intracellular sulfhydryl antioxidant and a scavenger of free radicals and thereby protect cellular membranes and other oxidation-prone structures in the cell from damage due to oxygen and oxygen-derived free radicals (55). It was shown that T4C stimulates oxygen uptake in rat liver mitochondria (43). As T4C plays an important role in oxidative-defense mechanisms (55), it was of interest to examine the effect of T4C and MT4C on the amount of intracellular ROS. Our previous study showed that when E. histolytica trophozoites were cultured under l-cysteine-limited conditions for 72 h, the intracellular levels of reactive oxygen species increased 4-fold (18). We cultivated trophozoites in l-cysteine-deprived BI-S-33 medium for 72 h, and the medium was replaced with l-cysteine-deprived BI-S-33 medium containing 2 mM T4C, MT4C, or l-cysteine. After 3 h, the relative level of ROS was measured using the fluorescent indicator CM-H 2DCFDA [5-(and-6)-chloromethyl-2′,7′-dichlorodihydrofluorescein diacetate, acetyl ester]. We found that the intracellular levels of reactive oxygen species in trophozoites cultured with 2 mM T4C, MT4C, or l-cysteine were, respectively, approximately 50, 21, or 32% lower than those in control cells (Fig. 6, bar Cys dep). These results suggest that T4C, M4C, and l-cysteine (T4C in particular) are important scavengers of reactive oxygen species in E. histolytica. The level of suppression of ROS by supplemented thiazolidine-4-carboxylic acids in the l-cysteine-deprived culture medium was only partial (<50%). This may be because, besides the thiazolidine-4-carboxylic acids described here, l-cysteine-derived metabolites that are involved in the antioxidant defense mechanism may exist in E. histolytica. Mackenzie and Harris were the first to recognize the therapeutic potential of T4C in animals (43). They noted that T4C is about five times more potent than l-cysteine in preventing massive pleural effusions and death in thiourea-treated rats. It was presumed that T4C, possessing a protected sulfur atom in its ring, opens and frees a sulfhydryl group after entering a liver cell. l-Cysteine, on the other hand, has an unprotected free sulfhydryl group, which is likely to react with oxidants before entering a cell.

Bottom Line: Furthermore, T4C and MT4C significantly enhanced trophozoite growth and reduced intracellular reactive oxygen species (ROS) levels when it was added to cultures, suggesting that 2-(R)-thiazolidine-4-carboxylic acids are involved in the defense against oxidative stress.We found that L-cysteine inside the cell rapidly reacts with aldehydes to form 2-(R)-thiazolidine-4-carboxylic acid.We showed that these 2-(R)-thiazolidine-4-carboxylic derivatives serve as an L-cysteine source, promote growth, and protect cells against oxidative stress by scavenging aldehydes and reducing the ROS level.

View Article: PubMed Central - PubMed

Affiliation: Department of Parasitology, National Institute of Infectious Diseases, Shinjuku, Tokyo, Japan.

Show MeSH
Related in: MedlinePlus