Schwer B, Lehman K, Saha N, Shuman S
Department of Microbiology and Immunology, Weill Medical College of Cornell University, Sloan-Kettering Institute, New York, New York 10021, USA.
J Biol Chem. 2001 Jan 19;276(3):1857-64. doi: 10.1074/jbc.M006072200. Epub 2000 Oct 16.
The mRNA capping apparatus of the pathogenic fungus Candida albicans consists of three components: a 520- amino acid RNA triphosphatase (CaCet1p), a 449-amino acid RNA guanylyltransferase (Cgt1p), and a 474-amino acid RNA (guanine-N7-)-methyltransferase (Ccm1p). The fungal guanylyltransferase and methyltransferase are structurally similar to their mammalian counterparts, whereas the fungal triphosphatase is mechanistically and structurally unrelated to the triphosphatase of mammals. Hence, the triphosphatase is an attractive antifungal target. Here we identify a biologically active C-terminal domain of CaCet1p from residues 202 to 520. We find that CaCet1p function in vivo requires the segment from residues 202 to 256 immediately flanking the catalytic domain from 257 to 520. Genetic suppression data implicate the essential flanking segment in the binding of CaCet1p to the fungal guanylyltransferase. Deletion analysis of the Candida guanylyltransferase demarcates an N-terminal domain, Cgt1(1-387)p, that suffices for catalytic activity in vitro and for cell growth. An even smaller domain, Cgt1(1-367)p, suffices for binding to the guanylyltransferase docking site on yeast RNA triphosphatase. Deletion analysis of the cap methyltransferase identifies a C-terminal domain, Ccm1(137-474)p, as being sufficient for cap methyltransferase function in vivo and in vitro. Ccm1(137-474)p binds in vitro to synthetic peptides comprising the phosphorylated C-terminal domain of the largest subunit of RNA polymerase II. Binding is enhanced when the C-terminal domain is phosphorylated on both Ser-2 and Ser-5 of the YSPTSPS heptad repeat. We show that the entire three-component Saccharomyces cerevisiae capping apparatus can be replaced by C. albicans enzymes. Isogenic yeast cells expressing "all-Candida" versus "all-mammalian" capping components can be used to screen for cytotoxic agents that specifically target the fungal capping enzymes.
一种含520个氨基酸的核糖核酸三磷酸酶（CaCet1p）、一种含449个氨基酸的核糖核酸鸟苷酸转移酶（Cgt1p）和一种含474个氨基酸的核糖核酸（鸟嘌呤 - N7 -）甲基转移酶（Ccm1p）。真菌的鸟苷酸转移酶和甲基转移酶在结构上与其哺乳动物对应物相似，而真菌的三磷酸酶在机制和结构上与哺乳动物的三磷酸酶无关。因此，三磷酸酶是一个有吸引力的抗真菌靶点。在这里，我们鉴定出CaCet1p从第202位到第520位残基的具有生物活性的C末端结构域。我们发现CaCet1p在体内发挥功能需要紧邻257至520位催化结构域的202至256位残基片段。基因抑制数据表明这个必需的侧翼片段参与CaCet1p与真菌鸟苷酸转移酶的结合。对念珠菌鸟苷酸转移酶的缺失分析划定了一个N末端结构域Cgt1(1 - 387)p，它在体外具有催化活性且对细胞生长而言是足够的。一个更小的结构域Cgt1(1 - 367)p足以与酵母核糖核酸三磷酸酶上的鸟苷酸转移酶对接位点结合。对帽甲基转移酶的缺失分析鉴定出一个C末端结构域Ccm1(137 - 474)p，它在体内和体外对帽甲基转移酶功能而言都是足够的。Ccm1(137 - 474)p在体外与包含核糖核酸聚合酶II最大亚基磷酸化C末端结构域的合成肽结合。当YSPTSPS七肽重复序列的Ser - 2和Ser - 5都被磷酸化时，结合会增强。我们表明酿酒酵母完整的三组分加帽装置可以被白色念珠菌的酶替代。表达“全白色念珠菌”与“全哺乳动物”加帽组分的同基因酵母细胞可用于筛选特异性靶向真菌加帽酶的细胞毒性剂。
