Data Sheet 9_Tetrahymena thermophila glutathione-S-transferase superfamily: an eco-paralogs gene network differentially responding to various environmental abiotic stressors and an update on this gene family in ciliates.pdf

Glutathione S-transferases constitute a superfamily of enzymes involved mainly, but not exclusively, in the detoxification of xenobiotic compounds that are considered environmental pollutants. In this work, an updated analysis of putative cytosolic glutathione S-transferases (cGST) from ciliate protozoa is performed although this analysis is mainly focused on Tetrahymena thermophila. Among ciliates, the genus Tetrahymena has the highest number (58 on average) of cGST genes. As in mammals, the Mu class of cGST is present in all analyzed ciliates and is the majority class in Tetrahymena species. After an analysis of the occurrence of GST domains in T. thermophila, out of the 54 GSTs previously considered to be Mu class, six of them have been discarded as they do not have recognizable GST domains. In addition, there is one GST species-specific and another GST-EF1G (elongation factor 1 gamma). A structural analysis of T. thermophila GSTs has shown a wide variety of β-sheets/α-helix patterns, one of the most abundant being the canonical thioredoxin-folding pattern. Within the categories of bZIP and C4 zinc finger transcription factors, potential binding sites for c-Jun and c-Fos are abundant (32% as average), along with GATA-1 (71% average) in the T. thermophila GST gene promoters. The alignment of all MAPEG (Membrane Associated Proteins involved in Eicosanoid and Glutathione metabolism) GST protein sequences from Tetrahymena species shows that this family is divided into two well-defined clans. The phylogenetic analysis of T. thermophila GSTs has shown that a cluster of 19 Mu-class GST genes are phylogenetic predecessors of members from the omega, theta and zeta classes. This means that the current GST phylogenetic model needs to be modified. Sixteen T. thermophila GST genes, together with two clusters including three genes each with very high identity, have been selected for qRT-PCR analysis under stress from eleven different environmental stressors. This analysis has revealed that there are GST genes that respond selectively and/or differentially to each stressor, independently of the GST class to which it belongs. Most of them respond to the two more toxic metal(loid)s used (Cd or As).

谷胱甘肽S-转移酶（Glutathione S-transferases, GST）是一类超家族酶，主要功能但不限于参与被视为环境污染物的外源性化合物的解毒过程。本研究对纤毛原生动物的推定胞质谷胱甘肽S-转移酶（cytosolic glutathione S-transferases, cGST）开展了更新式分析，研究重点主要聚焦于嗜热四膜虫（Tetrahymena thermophila）。在纤毛虫类群中，四膜虫属的cGST基因数量最多，平均可达58个。与哺乳动物类似，cGST的Mu类存在于所有被分析的纤毛虫中，且是四膜虫物种中的主要类别。通过对嗜热四膜虫GST结构域分布的分析，在此前被归类为Mu类的54个GST成员中，有6个因未携带可识别的GST结构域而被排除。此外，还存在1个物种特异性GST以及1个GST-EF1G（延伸因子1γ，elongation factor 1 gamma）。对嗜热四膜虫GST的结构分析显示，其β折叠/α螺旋模式具有丰富多样性，其中最常见的一类为经典硫氧还蛋白折叠模体。在嗜热四膜虫GST基因的启动子区域中，碱性亮氨酸拉链（bZIP）与C4型锌指转录因子类别的c-Jun和c-Fos潜在结合位点较为丰富（平均占比32%），GATA-1（GATA结合蛋白1）的潜在结合位点平均占比达71%。对四膜虫物种所有MAPEG（膜相关花生四烯酸与谷胱甘肽代谢蛋白，Membrane Associated Proteins involved in Eicosanoid and Glutathione metabolism）GST蛋白序列进行多序列比对后发现，该家族可分为两个界限明确的进化枝。对嗜热四膜虫GST的系统发育分析显示，包含19个Mu类GST基因的进化簇是Omega类、Theta类与Zeta类GST成员的系统发育前体，这表明当前的GST系统发育模型需要进行修正。研究选取了16个嗜热四膜虫GST基因，以及2个各含3个高度同源基因的基因簇，针对11种不同环境胁迫因子开展了实时定量聚合酶链反应（quantitative real-time polymerase chain reaction, qRT-PCR）分析。该分析结果显示，存在可对每种胁迫因子产生选择性和/或差异性响应的GST基因，且该响应与其所属的GST类别无关。其中大多数GST基因会对实验所用的两种毒性最强的金属及类金属——镉（Cd）与砷（As）产生响应。