Instituto de Biología Molecular y Celular de Rosario, CONICET, Facultad de Ciencias Bioquímicas y Farmacéuticas, Universidad Nacional de Rosario, Rosario, Argentina.
Mol Biol Evol. 2013 Jul;30(7):1630-43. doi: 10.1093/molbev/mst076. Epub 2013 Apr 19.
The ciliate Tetrahymena thermophila incorporates sterols from its environment that desaturates at positions C5(6), C7(8), and C22(23). Phytosterols are additionally modified by removal of the ethyl group at carbon 24 (C24). The enzymes involved are oxygen-, NAD(P)H-, and cytochrome b5 dependent, reason why they were classified as members of the hydroxylases/desaturases superfamily. The ciliate's genome revealed the presence of seven putative sterol desaturases belonging to this family, two of which we have previously characterized as the C24-de-ethylase and C5(6)-desaturase. A Rieske oxygenase was also identified; this type of enzyme, with sterol C7(8)-desaturase activity, was observed only in animals, called Neverland in insects and DAF-36 in nematodes. They perform the conversion of cholesterol into 7-dehydrocholesterol, first step in the synthesis of the essential hormones ecdysteroids and dafachronic acids. By adapting an RNA interference-by-feeding protocol, we easily screened six of the eight genes described earlier, allowing the characterization of the Rieske-like oxygenase as the ciliate's C7(8)-desaturase (Des7p). This characterization was confirmed by obtaining the corresponding knockout mutant, making Des7p the first nonanimal Rieske-sterol desaturase described. To our knowledge, this is the first time that the feeding-RNAi technique was successfully applied in T. thermophila, enabling to consider such methodology for future reverse genetics high-throughput screenings in this ciliate. Bioinformatics analyses revealed the presence of Des7p orthologs in other Oligohymenophorean ciliates and in nonanimal Opisthokonts, like the protists Salpingoeca rosetta and Capsaspora owczarzaki. A horizontal gene transfer event from a unicellular Opisthokont to an ancient phagotrophic Oligohymenophorean could explain the acquisition of the Rieske oxygenase by Tetrahymena.
嗜热四膜虫从环境中摄取固醇,这些固醇在 C5(6)、C7(8) 和 C22(23)位发生去饱和作用。植物固醇还通过去除 C24 位的乙基基团进一步修饰。涉及的酶依赖于氧、NAD(P)H 和细胞色素 b5,因此它们被归类为羟化酶/去饱和酶超家族的成员。纤毛虫的基因组揭示了存在七种属于该家族的推定固醇去饱和酶,其中两种我们之前已将其特征化为 C24-去乙基酶和 C5(6)-去饱和酶。还鉴定了一种 Rieske 加氧酶;这种具有固醇 C7(8)-去饱和酶活性的酶仅在动物中观察到,在昆虫中称为 Neverland,在线虫中称为 DAF-36。它们将胆固醇转化为 7-脱氢胆固醇,这是合成必需激素蜕皮甾酮和 dafachronic 酸的第一步。通过适应 RNA 干扰喂食方案,我们轻松筛选了前面描述的八个基因中的六个,从而将 Rieske 样加氧酶鉴定为纤毛虫的 C7(8)-去饱和酶(Des7p)。通过获得相应的敲除突变体,证实了这种特征,使 Des7p 成为第一个描述的非动物 Rieske-固醇去饱和酶。据我们所知,这是首次成功将喂食-RNAi 技术应用于嗜热四膜虫,使这种方法可以考虑用于未来该纤毛虫的反向遗传学高通量筛选。生物信息学分析表明,Des7p 的同源物存在于其他寡膜纤毛虫和非动物后生动物中,如原生动物玫瑰旋口虫和Capsaspora owczarzaki。从单细胞后生动物到古老的吞噬性寡膜纤毛虫的水平基因转移事件可以解释 Tetrahymena 获得 Rieske 加氧酶的原因。