Gagnon Dominic, Foucher Aude, Girard Isabelle, Ouellette Marc
Centre de Recherche en Infectiologie du Centre de recherche du CHUL, Division de Microbiologie, Faculté de Médecine, Université Laval, Québec, Canada.
Mol Biochem Parasitol. 2006 Nov;150(1):63-71. doi: 10.1016/j.molbiopara.2006.06.009. Epub 2006 Jul 13.
Serine hydroxymethyltransferase (SHMT) catalyses the reversible conversion of serine and tetrahydrofolate to glycine and methylene-tetrahydrofolate. The recent completion of the genome sequence of Leishmania major revealed the presence of two genes coding for two isoforms of this protein. In silico analysis showed that one isoform had an extension at its N-terminus and was predicted to localize to the mitochondrion. The situation is different in other kinetoplastid parasites with only one SHMT encoding gene in Trypanosoma cruzi and no SHMT encoding gene in Trypanosoma brucei. The two L. major SHMT genes were cloned in frame with the green fluorescent protein and the resulting fusion proteins showed differential localization: the short form (SHMT-S) was found in the cytosol while the long one (SHMT-L) was found in an organelle that has hallmarks of the parasite mitochondrion. Indeed, SHMT-L had a similar cellular fractionation pattern as the mitochondrial HSP60 as determined by digitonin fractionation. Both SHMT-S and SHMT-L genes were expressed preferentially in the amastigote stage of the parasite and the RNA levels of SHMT-L could be modulated by glycine, serine, and folate. Overexpression of SHMT-S increased resistance to the antifolate methotrexate and to a lower level to the inhibitor thiosemicarbazide in a rich folate containing medium. These findings suggest that folate metabolism is compartmentalised in Leishmania and that SHMT RNA levels are responsive to environmental conditions.
丝氨酸羟甲基转移酶（SHMT）催化丝氨酸和四氢叶酸可逆转化为甘氨酸和亚甲基四氢叶酸。利什曼原虫（Leishmania major）基因组序列最近完成，结果显示存在两个编码该蛋白两种同工型的基因。计算机分析表明，一种同工型在其N端有一个延伸，预计定位于线粒体。在其他动基体寄生虫中情况则不同，克氏锥虫（Trypanosoma cruzi）只有一个编码SHMT的基因，而布氏锥虫（Trypanosoma brucei）没有编码SHMT的基因。将利什曼原虫的两个SHMT基因与绿色荧光蛋白读框克隆，所得融合蛋白显示出不同的定位：短型（SHMT-S）存在于细胞质中，而长型（SHMT-L）存在于具有寄生虫线粒体特征的细胞器中。实际上，通过洋地黄皂苷分级分离确定，SHMT-L与线粒体热休克蛋白60（HSP60）具有相似的细胞分级模式。SHMT-S和SHMT-L基因均在寄生虫的无鞭毛体阶段优先表达，并且SHMT-L的RNA水平可受甘氨酸、丝氨酸和叶酸调节。在富含叶酸的培养基中，SHMT-S的过表达增加了对抗叶酸药物甲氨蝶呤的抗性，并对抑制剂氨基硫脲有较低水平的抗性。这些发现表明，利什曼原虫中的叶酸代谢是分区进行的，并且SHMT的RNA水平对环境条件有反应。
