• 主题
高级搜索  >
历史搜索 清空历史
首页> 外文学位 >Functional characterization of Leishmania infantum asparagine synthetase A and ribose 5-phosphate isomerase B as potential drug targets
【24h】

Functional characterization of Leishmania infantum asparagine synthetase A and ribose 5-phosphate isomerase B as potential drug targets

机译：婴儿利什曼原虫天冬酰胺合成酶A和核糖5-磷酸异构酶B作为潜在药物靶标的功能表征

获取原文
获取原文并翻译 | 示例
页面导航
  • 摘要
  • 著录项
  • 相似文献
  • 相关主题

摘要

Leishmaniasis is a neglected tropical disease caused by Leishmania parasites associated with an important burden worldwide. Visceral leishmaniasis (VL) is the most severe form of the disease, and it is fatal if left untreated. Disease control relies mostly on chemotherapy, which is frequently associated to safety issues, drug resistance, among other disadvantages that hinder disease eradication in endemic areas. Therefore, the search for new drugs and novel drug targets becomes imperative. In order to identify new potential molecular targets, an in silico comparative genomic analysis has been performed. Asparagine synthetase A (AS-A) and ribose-5-phosphate isomerase B (RPIB), which lack human homologues, have been selected as promising candidates. This thesis presents the first functional characterization of these two proteins in Leishmania infantum and Trypanosoma brucei, including both biochemical and genetic studies. Asparagine synthase (AS) is responsible for the conversion of aspartate into asparagine (Asn) in an ATP-dependent manner, using ammonia or glutamine as a nitrogen source. There are two structurally distinct AS: the strictly ammonia dependent, type A, and the type B, which preferably uses glutamine. Interestingly, we found that AS-A enzymes from trypanosomatids can use both ammonia and glutamine as nitrogen donors. Moreover, we have successfully generated ASA null mutants by targeted gene replacement in L. infantum and these parasites do not exhibit significant growth or infectivity defects. Indeed, a severe impairment of in vitro growth was only observed when null mutants were cultured in Asn limiting conditions. Our results demonstrate that despite being important upon Asn deprivation, LiAS-A is not essential for parasite survival, growth or infectivity in normal in vitro and in vivo conditions. The same pattern was observed in the related parasite T. brucei, using RNAi studies. Therefore, we exclude AS-A as a suitable drug target against trypanosomatids. Ribose-5-phosphate isomerase belongs to the non-oxidative branch of the pentose phosphate pathway, catalysing the interconversion of D-ribose-5-phosphate (R5P) and Dribulose- 5-phosphate (Ru5P). Trypanosomatids encode a type B RPI, whereas humans have a structurally unrelated type A. RPIB from trypanosomatids preferentially converts Ru5P into R5P. We demonstrated that its knockdown in T. brucei dramatically impairs parasites infectivity. Moreover, null mutants generation in L. infantum was only possible when an episomal copy of RPIB gene was provided, and the latter was preserved both in vitro and in vivo, even in the absence of drug pressure for a long period indicating the gene is essential for survival. In vitro, sKO promastigotes exhibited no defect in growth, metacyclogenesis or macrophages infection, however, an impairment in intracellular amastigotes' replication was observed. Additionally, mice infected with LisKO mutants presented a reduced parasite burden in the liver, rescued by RPIB complementation. To gain further insights whether RPIB essentiality is due to its isomerase function, sKO mutants were complemented with an episomal copy of RPIB carrying a mutation on Cys69 that abrogates isomerase function. In this case, the inability to remove the second allele of RPIB gene suggests the essentiality is due to the annotated metabolic function. Moreover, T. brucei is reluctant to complete RPIB removal, and mice experienced an extended survival upon infection with sKO mutants. In summary, our results genetically validate RPIB as a novel drug target candidate in trypanosomatids.
机译：利什曼病是一种由利什曼原虫引起的被忽视的热带病，与世界范围内的重要负担有关。内脏利什曼病（VL）是该病的最严重形式，如果不治疗会致命。疾病控制主要依靠化学疗法，化学疗法通常与安全性问题，耐药性相关，以及阻碍在流行地区根除疾病的其他缺点。因此，寻找新药和新药靶标成为当务之急。为了鉴定新的潜在分子靶标，已经进行了计算机模拟比较基因组分析。缺乏人同源性的天冬酰胺合成酶A（AS-A）和5核糖核糖异构酶B（RPIB）已被选为有前途的候选药物。本文介绍了这两种蛋白质在婴儿利什曼原虫和布鲁氏锥虫中的第一个功能特征，包括生化和遗传研究。天冬酰胺合成酶（AS）负责使用氨或谷氨酰胺作为氮源，以ATP依赖性方式将天冬氨酸转化为天冬酰胺（Asn）。在结构上有两种不同的AS：严格依赖氨的A型和B型，最好使用谷氨酰胺。有趣的是，我们发现锥虫的AS-A酶可以同时使用氨和谷氨酰胺作为氮供体。此外，我们已经通过在婴儿乳杆菌中的靶向基因置换成功地产生了ASA null突变体，这些寄生虫没有显示出明显的生长或感染性缺陷。实际上，仅当在Asn限制条件下培养无效突变体时，才观察到严重的体外生长损伤。我们的结果表明，尽管在Asn剥夺中很重要，但LiAS-A对于正常的体外和体内条件下的寄生虫存活，生长或感染性并不是必不可少的。使用RNAi研究，在相关的寄生虫布鲁氏菌中观察到了相同的模式。因此，我们排除AS-A作为抗锥虫病的合适药物靶标。 5磷酸核糖异构酶属于戊糖磷酸途径的非氧化分支，催化D-核糖5磷酸（R5P）和5磷酸核糖（Ru5P）的相互转化。锥虫编码B型RPI，而人类具有结构上无关的A型。锥虫的RPIB优先将Ru5P转化为R5P。我们证明了其在布鲁氏杆菌中的击倒极大地损害了寄生虫的传染性。此外，只有在提供了RPIB基因的游离复制后，才可能在婴儿乳杆菌中产生无效突变体，并且即使在长时间不存在药物压力的情况下，后者也可以在体内和体外保存，这表明该基因是必需的。为了生存。在体外，sKO前鞭毛体在生长，成环发生或巨噬细胞感染上均未表现出缺陷，但是，观察到胞内变形体复制受到损害。此外，感染了LisKO突变体的小鼠肝脏中的寄生虫负担减少，这是通过RPIB互补挽救的。为了进一步了解RPIB的必要性是否归因于其异构酶功能，在sKO突变体上附加了RPIB的附加型拷贝，该拷贝具有在Cys69上消除了异构酶功能的突变。在这种情况下，无法去除RPIB基因的第二个等位基因表明必要性是由于注释的代谢功能。此外，布鲁氏杆菌不愿完全去除RPIB，小鼠感染sKO突变体后存活期延长。总而言之，我们的研究结果从基因上证明了RPIB是锥虫病中新型的药物靶候选物。

著录项

  • 作者

    Faria, Joana Raquel Correia.;

  • 作者单位

    Universidade do Porto (Portugal).;

  • 授予单位 Universidade do Porto (Portugal).;
  • 学科 Pharmaceutical sciences.
  • 学位 Ph.D.
  • 年度 2016
  • 页码 331 p.
  • 总页数 331
  • 原文格式 PDF
  • 正文语种 eng
  • 中图分类
  • 关键词

相似文献

  • 外文文献
  • 中文文献
  • 专利
获取原文

客服邮箱：kefu@zhangqiaokeyan.com

京公网安备：11010802029741号 ICP备案号：京ICP备15016152号-6 六维联合信息科技 (北京) 有限公司©版权所有

  • 客服微信

  • 服务号