Niño Carlos H, Forero-Baena Nicolás, Contreras Luis E, Sánchez-Lancheros Diana, Figarella Katherine, Ramírez María H
Laboratorio de Investigaciones Básicas en Bioquímica, Facultad de Ciencias, Universidad Nacional de Colombia, Bogotá, Colombia.
Fundación Instituto de Estudios Avanzados, Caracas, Venezuela.
Mem Inst Oswaldo Cruz. 2015 Nov;110(7):890-7. doi: 10.1590/0074-02760150175.
The intracellular parasite Trypanosoma cruzi is the aetiological agent of Chagas disease, a public health concern with an increasing incidence rate. This increase is due, among other reasons, to the parasite's drug resistance mechanisms, which require nicotinamide adenine dinucleotide (NAD+). Furthermore, this molecule is involved in metabolic and intracellular signalling processes necessary for the survival of T. cruzi throughout its life cycle. NAD+biosynthesis is performed by de novo and salvage pathways, which converge on the step that is catalysed by the enzyme nicotinamide mononucleotide adenylyltransferase (NMNAT) (enzyme commission number: 2.7.7.1). The identification of the NMNAT of T. cruzi is important for the development of future therapeutic strategies to treat Chagas disease. In this study, a hypothetical open reading frame (ORF) for NMNAT was identified in the genome of T. cruzi.The corresponding putative protein was analysed by simulating structural models. The ORF was amplified from genomic DNA by polymerase chain reaction and was further used for the construction of a corresponding recombinant expression vector. The expressed recombinant protein was partially purified and its activity was evaluated using enzymatic assays. These results comprise the first identification of an NMNAT in T. cruzi using bioinformatics and experimental tools and hence represent the first step to understanding NAD+ metabolism in these parasites.
细胞内寄生虫克氏锥虫是恰加斯病的病原体，恰加斯病是一个公共卫生问题，其发病率正在上升。这种上升除其他原因外，还归因于该寄生虫的耐药机制，而这些机制需要烟酰胺腺嘌呤二核苷酸（NAD+）。此外，该分子参与了克氏锥虫整个生命周期生存所必需的代谢和细胞内信号传导过程。NAD+的生物合成通过从头合成途径和补救途径进行，这两条途径在由烟酰胺单核苷酸腺苷酸转移酶（NMNAT）（酶学委员会编号：2.7.7.1）催化的步骤上汇聚。鉴定克氏锥虫的NMNAT对于开发未来治疗恰加斯病的治疗策略很重要。在本研究中，在克氏锥虫的基因组中鉴定出了一个NMNAT的假设开放阅读框（ORF）。通过模拟结构模型对相应的推定蛋白进行了分析。通过聚合酶链反应从基因组DNA中扩增出该ORF，并进一步用于构建相应的重组表达载体。对表达的重组蛋白进行了部分纯化，并使用酶促测定法评估了其活性。这些结果是首次使用生物信息学和实验工具在克氏锥虫中鉴定出NMNAT，因此代表了了解这些寄生虫中NAD+代谢的第一步。
