Huynh Tu T, Huynh Van T, Harmon Margaret A, Phillips Margaret A
Department of Pharmacology, University of Texas Southwestern Medical Center, Dallas, Texas 75390-9041, USA.
J Biol Chem. 2003 Oct 10;278(41):39794-800. doi: 10.1074/jbc.M306306200. Epub 2003 Jul 29.
The parasitic protozoa Trypanosoma brucei utilizes a novel cofactor (trypanothione, T(SH)2), which is a conjugate of GSH and spermidine, to maintain cellular redox balance. gamma-Glutamylcysteine synthetase (gamma-GCS) catalyzes the first step in the biosynthesis of GSH. To evaluate the importance of thiol metabolism to the parasite, RNAi methods were used to knock down gene expression of gamma-GCS in procyclic T. brucei cells. Induction of gamma-GCS RNAi with tetracycline led to cell death within 4-6 days post-induction. Cell death was preceded by the depletion of the gamma-GCS protein and RNA and by the loss of the cellular pools of GSH and T(SH)2. The addition of GSH (80 microM) to cell cultures rescued the RNAi cell death phenotype and restored the intracellular thiol pools to wild-type levels. Treatment of cells with buthionine sulfoximine (BSO), an enzyme-activated inhibitor of gamma-GCS, also resulted in cell death. However, the toxicity of the inhibitor was not reversed by GSH, suggesting that BSO has more than one cellular target. BSO depletes intracellular thiols to a similar extent as gamma-GCS RNAi; however, addition of GSH did not restore the pools of GSH and T(SH)2. These data suggest that BSO also acts to inhibit the transport of GSH or its peptide metabolites into the cell. The ability of BSO to inhibit both synthesis and transport of GSH likely makes it a more effective cytotoxic agent than an inhibitor with a single mode of action. Finally the potential for the T(SH)2 biosynthetic enzymes to be regulated in response to reduced thiol levels was studied. The expression levels of ornithine decarboxylase and of S-adenosylmethionine decarboxylase, two essential enzymes in spermidine biosynthesis, remained constant in induced gamma-GCS RNAi cell lines.
寄生原生动物布氏锥虫利用一种新型辅因子（锥虫硫醇，T(SH)2）来维持细胞氧化还原平衡，锥虫硫醇是谷胱甘肽（GSH）和亚精胺的共轭物。γ-谷氨酰半胱氨酸合成酶（γ-GCS）催化GSH生物合成的第一步。为了评估硫醇代谢对该寄生虫的重要性，采用RNA干扰方法敲低前循环期布氏锥虫细胞中γ-GCS的基因表达。用四环素诱导γ-GCS RNA干扰导致诱导后4 - 6天内细胞死亡。细胞死亡之前，γ-GCS蛋白和RNA耗竭，细胞内GSH和T(SH)2池丧失。向细胞培养物中添加GSH（80微摩尔）挽救了RNA干扰细胞死亡表型，并使细胞内硫醇池恢复到野生型水平。用丁硫氨酸亚砜胺（BSO）处理细胞，BSO是γ-GCS的酶激活抑制剂，也导致细胞死亡。然而，GSH不能逆转该抑制剂的毒性，这表明BSO有多个细胞靶点。BSO使细胞内硫醇耗竭的程度与γ-GCS RNA干扰相似；然而，添加GSH并未恢复GSH和T(SH)2池。这些数据表明，BSO还可抑制GSH或其肽代谢产物转运进入细胞。BSO抑制GSH合成和转运的能力可能使其成为比具有单一作用模式的抑制剂更有效的细胞毒性剂。最后，研究了锥虫硫醇生物合成酶响应硫醇水平降低而被调节的可能性。在诱导γ-GCS RNA干扰的细胞系中，亚精胺生物合成中的两种关键酶鸟氨酸脱羧酶和S-腺苷甲硫氨酸脱羧酶的表达水平保持恒定。
