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首页> 外文学位 >The characterization of Plasmodium falciparum iron regulatory-like protein (PfIRPa) in asexual stage Plasmodium falciparum parasites.
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The characterization of Plasmodium falciparum iron regulatory-like protein (PfIRPa) in asexual stage Plasmodium falciparum parasites.

机译：无性疟原虫恶性疟原虫中恶性疟原虫铁调节样蛋白（PfIRPa）的表征。

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摘要

Iron is an indispensable nutrient for the erythrocytic forms of Plasmodium falciparum, but the metabolic pathways of iron homeostasis in Plasmodium falciparum are poorly understood. An iron regulatory-like protein expressed by Plasmodium falciparum (PfIRPa) is a plausible candidate for having a role in iron homeostasis of asexual Plasmodium falciparum parasites. This protein has homology to mammalian iron regulatory proteins/aconitases, and is capable of binding RNA iron response elements (IREs). Western blot analysis of digitonin-permeabilized trophozoites suggests that the localization of PfIRPa is predominantly in membranous compartments of the parasite, such as the mitochondrion. However, immunofluorescence analysis shows that PfIRPa is localized in the mitochondria and the parasitic cytosol. Under conditions favoring iron-sulfur cluster formation, recombinant PfIRPa (rPfIRPa) displays aconitase activity, as detected by a colorimetric NADPH-MTT (3-[4.5-dimethylthiazol-2,5-diphenyl tetrazolium bromide) assay. Spectrophotometric measurements of cis-aconitate hydration at 240 nm show that rPfIRPa had an overall catalytic efficiency (Kcat/Km) that was similar in magnitude to human cytosolic IRP1/aconitase and human mitochondrial aconitase. PflRPa immunoprecipitated from parasite lysates also has aconitase activity, as assessed by an MTT-based assay. PfIRPa binds specifically to an IRE-like stem loop structure found in the 3' untranslated region of malarial gene PF13_0080. The location of this IRE is consistent with the possibility that PfIRPa could protect PF13_0080 mRNA from endonucleolytic cleavage by binding to this IRE-like structure. To test this hypothesis, two small (25-mer) RNA-mimicking oligonucleotides were designed: one identical and one complementary to the stem-loop structure of PF13_0080 IRE. Contrary to expectations, the addition of either oligonucleotide to erythrocytic Plasmodium falciparum led to increased levels of PF13_0080 mRNA, decreased levels of PfIRPa mRNA, and sustained malaria growth despite substrate limitations caused by high parasitemia culturing conditions. The results presented in this work provide evidence that PfIRPa localizes in the mitochondrion and in the cytosol and is able to demonstrate aconitase activity. The results also suggest that PfIRPa might act as a post-transcriptional repressor of PF13_0080 mRNA. Further understanding of the role of PfIRPa/aconitase in the regulation of Plasmodium falciparum iron homeostasis may contribute towards the development of novel antimalarial strategies against plasmodial species.
机译：铁是恶性疟原虫红细胞形式必不可少的营养素，但恶性疟原虫中铁稳态的代谢途径知之甚少。由恶性疟原虫（PfIRPa）表达的铁调节样蛋白是在无性恶性疟原虫寄生虫的铁稳态中起作用的合理候选者。该蛋白与哺乳动物的铁调节蛋白/ aconitases具有同源性，并且能够结合RNA铁反应元件（IREs）。洋地黄素透化滋养体的蛋白质印迹分析表明，PfIRPa的定位主要位于寄生虫的膜区室，例如线粒体。但是，免疫荧光分析表明PfIRPa位于线粒体和寄生细胞质中。在有利于铁硫簇形成的条件下，重组PfIRPa（rPfIRPa）表现出乌头酸酶活性，通过比色NADPH-MTT（3- [4.5-二甲基噻唑-2,5-二苯基溴化四氮唑]测定）检测。分光光度法在240 nm处测定顺式衣康酸水合的结果表明，rPfIRPa的总催化效率（Kcat / Km）大小与人胞质IRP1 / aconitase和人线粒体乌头酸酶相似。如通过基于MTT的分析所评估，从寄生虫裂解物中免疫沉淀的PflRPa也具有乌头酸酶活性。 PfIRPa特异性结合在疟疾基因PF13_0080的3'非翻译区中发现的IRE类茎环结构。此IRE的位置与PfIRPa可以通过与该IRE-like结构结合来保护PF13_0080 mRNA免受内切核酸酶裂解的可能性相一致。为了验证这一假设，设计了两个小的（25-mer）RNA模仿寡核苷酸：一个与PF13_0080 IRE的茎环结构相同，另一个与之互补。与预期相反，尽管由高寄生性细菌培养条件引起的底物限制，但在促红细胞性恶性疟原虫中添加任一种寡核苷酸均导致PF13_0080 mRNA水平升高，PfIRPa mRNA水平降低以及持续的疟疾生长。这项工作中提出的结果提供了证据，证明PfIRPa定位于线粒体和细胞质中，并且能够证明乌头酸酶的活性。结果还表明，PfIRPa可能充当PF13_0080 mRNA的转录后阻遏物。进一步了解PfIRPa / aconitase在恶性疟原虫铁稳态调节中的作用可能有助于开发针对疟原虫物种的新型抗疟疾策略。

著录项

  • 作者

    Hodges, Marcus G.;

  • 作者单位

    Howard University.;

  • 授予单位 Howard University.;
  • 学科 Biology Molecular.; Biology Microbiology.; Health Sciences Pathology.
  • 学位 Ph.D.
  • 年度 2005
  • 页码 105 p.
  • 总页数 105
  • 原文格式 PDF
  • 正文语种 eng
  • 中图分类 分子遗传学;微生物学;病理学;
  • 关键词

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