BIOCHEMISTRY OF SCHISTOSOME GLUTATHIONE S-TRANSFERASES

据估计，血吸虫病困扰着2亿多人。 全世界。在实用的疫苗问世之前，化疗将 对于治疗感染者和 旨在控制感染传播的计划 人口。因为临床上只有三种药物可供使用， 耐药性的出现仍令人担忧。 血吸虫与药物相互作用的生化基础的阐明 对于理解这两种药物的分子机制很重要 疗效和耐药性。曼氏血吸虫成虫在 至少六种生化上截然不同的谷胱甘肽S转移酶 (EC 2.5.1.18)已被证明在细胞内发挥作用 通过催化谷胱甘肽与A的偶联来保护 结构多样化的外源化合物和内源产生的 亲电者。尽管少数编码血吸虫谷胱甘肽的cDNA S转移酶已被分离并在细菌中过表达 产生候选疫苗抗原，目前还不知道这是如何实现的 重要的酶家族在寄生虫体内受到调节。我们有 发现曼氏血吸虫成虫暴露于三种原型 诱导剂(2(3)-叔丁基-4-羟基苯甲醚，3-甲基胆蒽， 和苯巴比妥)提高了mRNA的稳态水平， 与三种曼氏链球菌谷胱甘肽S转移酶之一杂交 类特异性cDNAs探针。这一观察表明， 这些基因中的一部分受到监管机制的影响，这使得 寄生虫对化学刺激的反应，可能包括 抗血吸虫药物，在其环境中遇到。这项建议 旨在检验这一假设。具体的实验目标 包括：1)调查有阶段和性别选择性的 谷胱甘肽S转移酶基因的Northern杂交分析及 高压液相色谱；2)测定 2，3，7，8-四氯二苯并对二恶英和奥替拉唑诱导曼氏葡萄球菌 Northern杂交技术在谷胱甘肽S转移酶基因表达中的应用 分析特定的mRNA水平；3)利用核“跑跑式” 信使核糖核酸周转率的分析和测量，检验以下假设 黄曲霉毒素诱导血吸虫谷胱甘肽S转移酶基因表达 原型诱导剂受转录调控；以及4) 确定可能的调控序列(外源反应元件) 曼氏链霉菌谷胱甘肽S转移酶的5‘侧翼区 转录所需的同工酶亚基基因 激活。后一项实验将需要建造和 谷胱甘肽S-转移酶-萤火虫荧光素酶融合基因的表达 在真核细胞系中。

Schistosomiasis is estimated to afflict over 200 million people worldwide. Until a practical vaccine is available, chemotherapy will continue to be important both for treating infected individuals and in programs aimed at controlling infection transmission within populations. Because only three drugs are available for clinical use, the appearance of drug resistance is of continuing concern. Elucidation of the biochemical basis of schistosome-drug interactions is important for understanding the molecular mechanisms of both drug efficacy and drug resistance. Adult Schistosoma mansoni express at least six biochemically distinct forms of glutathione S-transferases (EC 2.5.1.18) that have been shown to play a role in cellular protection by catalyzing the conjugation of glutathione with a structurally diverse group of xenobiotics and endogenously produced electrophiles. Although a few cDNAs encoding schistosome glutathione S-transferases have been isolated and overexpressed in bacteria to produce candidate vaccine antigens, nothing is known about how this important family of enzymes is regulated within the parasite. We have discovered that exposure of adult S. mansoni to three prototypic inducing agents (2(3)-t-butyl-4-hydroxyanisole, 3-methylcholanthrene, and phenobarbital) elevates the steady-state level of mRNA that hybridizes to one of three S. mansoni glutathione S-transferase class-specific cDNA probes. This observation suggests that expression of those genes is subject to regulatory mecbanisms that allow the parasite to respond to chemical stimuli, including perhaps antischistosomal drugs, encountered in its environment. This proposal is designed to test that hypothesis. Specific experimental objectives include: 1) Survey the stage- and gender-selective expression of glutathione S-transferase genes by Northern hybridization analysis and high pressure liquid chromatography; 2) Determine whether 2,3,7,8-tetrachlordibenzo-p-dioxin and oltipraz induce S. mansoni glutathione S-transferase gene expression using Northern hybridization analysis to quantitate specific mRNA levels; 3) Using nuclear "run-on" assays and measurements of mRNA turnover, test the hypothesis that induction of schistosome glutathione S-transferase gene expression by prototypic inducing agents is transcriptionally regulated; and 4) Identify putative regulatory sequences (xenobiotic responsive elements) in the 5'-flanking region of a S. mansoni glutathione S-transferase isoenzyme subunit gene that are required for transcriptional activation. The latter experiments will entail construction and expression of glutathione S-transferase-firefly luciferase fusion genes in eukaryotic cell lines.