Characterization of a Novel Endoplasmic Reticulum Protein Involved in Tubercidin Resistance in Leishmania major
Characterization of a Novel Endoplasmic Reticulum Protein Involved in Tubercidin Resistance in Leishmania major
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DOI：
10.1371/journal.pntd.0004972
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发表时间：
2016-09-01
影响因子：
3.8
通讯作者：
Cotrim, Paulo Cesar
中科院分区：
文献类型：
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作者：
Aoki, Juliana Ide;Coelho, Adriano Cappellazzo;Cotrim, Paulo Cesar
研究背景利什曼原虫素（TUB）是一种具有潜在抗利什曼原虫活性的腺苷类似物，其作用机制和耐药性尚不完全清楚。为了了解作用机制和确定受这种药物影响的潜在代谢途径，我们在这项研究中采用了一种使用TUB的过表达/选择方法，用于确定潜在的靶点，以及L.少校虽然TUB对哺乳动物宿主具有毒性，但这些发现为基于嘌呤途径的合理抗利什曼病药物设计提供了依据。主要的Friedlin（LmjF）寄生虫和过表达/选择方法的应用，我们鉴定了两种含有能够赋予显著水平的TUB抗性的两个不同基因座的cosTU 1和cosTU 2。在cosTUB 1中含有编码NUPM 1样蛋白的基因，该蛋白先前已被描述为与L.亚马逊河。在cosTUB 2中，我们鉴定并表征了编码63 kDa蛋白的基因，我们将其表示为结核菌素抗性蛋白（TRP）。功能分析显示，转染子对TUB的敏感性低于LmjF寄生虫或用对照载体转染的那些。此外，cosTUB 2转染子中的trp mRNA和蛋白水平高于LmjF。TRP的免疫定位显示，它是共定位到内质网（ER），一个具有许多功能的细胞隔室。计算机模拟预测表明，TRP只包含一个假设的跨膜结构域。因此，这是可能的，TRP是一种管腔蛋白参与多药外排转运，可能参与嘌呤代谢pathway.Conclusions/Significance这项研究首次证明，TRP与TUB抗性利什曼原虫。下一个挑战是确定TRP如何介导TUB抗性以及嘌呤代谢是否受寄生虫中这种蛋白质的影响。最后，这些发现可能有助于开发针对嘌呤途径的替代抗利什曼原虫药物。
BackgroundTubercidin (TUB) is a toxic adenosine analog with potential antiparasitic activity against Leishmania, with mechanism of action and resistance that are not completely understood. For understanding the mechanisms of action and identifying the potential metabolic pathways affected by this drug, we employed in this study an overexpression/selection approach using TUB for the identification of potential targets, as well as, drug resistance genes in L. major. Although, TUB is toxic to the mammalian host, these findings can provide evidences for a rational drug design based on purine pathway against leishmaniasis.Methodology/Principal findingsAfter transfection of a cosmid genomic library into L. major Friedlin (LmjF) parasites and application of the overexpression/selection method, we identified two cosmids (cosTUB1 and cosTU2) containing two different loci capable of conferring significant levels of TUB resistance. In the cosTUB1 contained a gene encoding NUPM1-like protein, which has been previously described as associated with TUB resistance in L. amazonensis. In the cosTUB2 we identified and characterized a gene encoding a 63 kDa protein that we denoted as tubercidin-resistance protein (TRP). Functional analysis revealed that the transfectants were less susceptible to TUB than LmjF parasites or those transfected with the control vector. In addition, the trp mRNA and protein levels in cosTUB2 transfectants were higher than LmjF. TRP immunolocalization revealed that it was co-localized to the endoplasmic reticulum (ER), a cellular compartment with many functions. In silico predictions indicated that TRP contains only a hypothetical transmembrane domain. Thus, it is likely that TRP is a lumen protein involved in multidrug efflux transport that may be involved in the purine metabolic pathway.Conclusions/SignificanceThis study demonstrated for the first time that TRP is associated with TUB resistance in Leishmania. The next challenge is to determine how TRP mediates TUB resistance and whether purine metabolism is affected by this protein in the parasite. Finally, these findings may be helpful for the development of alternative anti-leishmanial drugs that target purine pathway.