Phelouzat M A, Lawrence F, Moulay L, Borot C, Schaeverbeke J, Schaeverbeke M, Robert-Gero M
Institut de Chimie des Substances Naturelles, CNRS-91198, Gif Sur Yvette, France.
Exp Parasitol. 1992 Mar;74(2):177-87. doi: 10.1016/0014-4894(92)90045-c.
Sinefungin, an antifungal and antiparasitic nucleoside antibiotic, is a very potent antileishmanial agent in vitro and in vivo (Bachrach et al. 1980, FEBS Letters 121, 287-291; Neal et al. 1985, Transactions of the Royal Society of Tropical Medicine and Hygiene 79, 85-122). It was previously shown that this molecule is a competitive inhibitor of AdoMet for transmethylases (Paolantonacci et al. 1986, Molecular and Biochemical Parasitology 21, 47-54; Avila et al. 1987, Molecular and Biochemical Parasitology 26, 69-76) and that it induces shape changes of Leishmania donovani promastigotes as observed by light microscopy (Lawrence and Robert-Gero 1990; Bulletin de la Societé Française de Parasitologie 8, 13-18). In the present work the effect of the antibiotic on the ultrastructure was analyzed by electron microscopy. The main changes induced at sublethal concentrations (0.26 microM sinefungin for 16 hr) were progressive rounding, decreased motility, enlargement of the flagellar pocket, and shortening and loss of the external part of the flagellum. The comparison with control cells showed shorter Golgi saccules and fragmentation of the trans-Golgi network into vesicles, indicating a stimulated Golgi apparatus activity. This result, associated with the enlarged flagellar pocket, suggests an unbalanced cytoplasmic exchange between exocytosis and endocytosis. These effects are quite different from those induced by tunicamycin (Dagger et al. 1984, Biology of the Cell 50; 173-180) or paromomycin. In addition, other nucleoside and nonnucleoside growth inhibitors failed to induce similar changes. AdoMet antagonized the sinefungin-induced shape changes and ultrastructural modifications but had no effect with respect to other growth inhibitors. This suggests that the sinefungin activity at the cellular level is specifically related to competition with AdoMet. A comparative study of N-methylation and carboxylmethylation of proteins in sinefungin-treated promastigotes showed that the antibiotic preferentially inhibits the latter, catalyzed by protein-O-methyltransferases. These enzymes are known to regulate the function of various proteins involved in secretion. Overall the results suggest that one of the main targets of sinefungin in exponentially growing cells is the protein carboxylmethylation involved in membrane transport.
西奈芬净是一种抗真菌和抗寄生虫的核苷类抗生素，在体外和体内都是一种非常有效的抗利什曼原虫剂（巴赫拉赫等人，1980年，《欧洲生物化学学会联合会快报》121卷，287 - 291页；尼尔等人，1985年，《皇家热带医学与卫生学会学报》79卷，85 - 122页）。先前的研究表明，该分子是S -腺苷甲硫氨酸（AdoMet）对甲基转移酶的竞争性抑制剂（保兰托纳奇等人，1986年，《分子与生化寄生虫学》21卷，47 - 54页；阿维拉等人，1987年，《分子与生化寄生虫学》26卷，69 - 76页），并且通过光学显微镜观察发现它会诱导杜氏利什曼原虫前鞭毛体的形态变化（劳伦斯和罗伯特 - 杰罗，1990年；《法国寄生虫学会通报》8卷，13 - 18页）。在本研究中，通过电子显微镜分析了该抗生素对超微结构的影响。在亚致死浓度（0.26微摩尔西奈芬净处理16小时）下诱导的主要变化是细胞逐渐变圆、运动性降低、鞭毛囊扩大、鞭毛外部缩短并消失。与对照细胞相比，高尔基体囊泡变短，反式高尔基体网络破碎成小泡，这表明高尔基体活性增强。这一结果与扩大的鞭毛囊相关，表明胞吐作用和胞吞作用之间的细胞质交换失衡。这些效应与衣霉素（达格尔等人，1984年，《细胞生物学》50卷；173 - 180页）或巴龙霉素诱导的效应有很大不同。此外，其他核苷类和非核苷类生长抑制剂未能诱导类似变化。AdoMet拮抗西奈芬净诱导的形态变化和超微结构改变，但对其他生长抑制剂没有影响。这表明西奈芬净在细胞水平的活性与和AdoMet的竞争特异性相关。对西奈芬净处理的前鞭毛体中蛋白质的N -甲基化和羧甲基化的比较研究表明，该抗生素优先抑制由蛋白质O -甲基转移酶催化的后者。已知这些酶调节参与分泌的各种蛋白质的功能。总体而言，结果表明西奈芬净在指数生长细胞中的主要靶点之一是参与膜转运的蛋白质羧甲基化。
