Instituto de Parasitología y Biomedicina López-Neyra, Consejo Superior de Investigaciones Científicas, Avda. del Conocimiento s/n, Armilla, Granada, Spain.
Int J Parasitol. 2012 Oct;42(11):975-89. doi: 10.1016/j.ijpara.2012.07.012. Epub 2012 Aug 29.
Sterol biosynthesis inhibitors are promising entities for the treatment of trypanosomal diseases. Insect forms of Trypanosoma brucei, the causative agent of sleeping sickness, synthesize ergosterol and other 24-alkylated sterols, yet also incorporate cholesterol from the medium. While sterol function has been investigated by pharmacological manipulation of sterol biosynthesis, molecular mechanisms by which endogenous sterols influence cellular processes remain largely unknown in trypanosomes. Here we analyse by RNA interference, the effects of a perturbation of three specific steps of endogenous sterol biosynthesis in order to dissect the role of specific intermediates in proliferation, mitochondrial function and cellular morphology in procyclic cells. A decrease in the levels of squalene synthase and squalene epoxidase resulted in a depletion of cellular sterol intermediates and end products, impaired cell growth and led to aberrant morphologies, DNA fragmentation and a profound modification of mitochondrial structure and function. In contrast, cells deficient in sterol methyl transferase, the enzyme involved in 24-alkylation, exhibited a normal growth phenotype in spite of a complete abolition of the synthesis and content of 24-alkyl sterols. Thus, the data provided indicates that while the depletion of squalene and post-squalene endogenous sterol metabolites results in profound cellular defects, bulk 24-alkyl sterols are not strictly required to support growth in insect forms of T. brucei in vitro.
甾醇生物合成抑制剂是治疗锥虫病的有前途的实体。昏睡病的病原体布氏锥虫的昆虫形式合成麦角固醇和其他 24-烷基化甾醇，但也从培养基中摄取胆固醇。尽管甾醇功能已通过甾醇生物合成的药理学操作进行了研究，但内源性甾醇影响细胞过程的分子机制在锥虫中仍知之甚少。在这里，我们通过 RNA 干扰分析了内源性甾醇生物合成的三个特定步骤受到干扰的影响，以便在裂殖体细胞中剖析特定中间体在增殖、线粒体功能和细胞形态中的作用。鲨烯合酶和鲨烯环氧化酶水平的降低导致细胞甾醇中间产物和终产物耗尽，细胞生长受损，并导致形态异常、DNA 片段化以及线粒体结构和功能的深刻改变。相比之下，尽管完全消除了 24-烷基化甾醇的合成和含量，但参与 24-烷基转移的酶甾醇甲基转移酶缺陷的细胞表现出正常的生长表型。因此，提供的数据表明，尽管 squalene 和 post-squalene 内源性甾醇代谢物的耗尽会导致严重的细胞缺陷，但在体外昆虫形式的 T. brucei 中，大量 24-烷基甾醇并不是支持生长所必需的。
