Proteome mapping of the protozoan parasite Leishmania and application to the study of drug targets and resistance mechanisms
Proteome mapping of the protozoan parasite Leishmania and application to the study of drug targets and resistance mechanisms
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DOI：
10.1074/mcp.m200085-mcp200
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发表时间：
2003-03-01
影响因子：
7
通讯作者：
Ouellette, M
中科院分区：
文献类型：
--
作者：
Drummelsmith, J;Brochu, V;Ouellette, M
利什曼原虫是一种原生动物寄生虫，在世界范围内造成严重的发病率和死亡率。到目前为止，很少有寄生虫受到蛋白质组分析，但主要利什曼原虫的基因组测序项目目前正在进行中，使这些研究成为可能。在这里，我们提出了一个高分辨率的蛋白质组，包括近3700个点，使其成为最完整的二维凝胶表达的寄生虫蛋白质组产生的日期。我们已经通过质谱学鉴定了一些标志性蛋白，并表明其中一些对相关物种杜氏利什曼原虫幼虫是有效的。我们还观察到了α-微管蛋白和β-微管蛋白的几种形式和片段，并表明这些片段的数量和数量随着寄生虫培养的年龄而增加。锥体硫酮还原酶(TRYR)是锥虫体内谷胱甘肽还原酶的替代物，是这些寄生虫所特有的一种必需蛋白质，因此作为药物靶标受到了广泛的关注。对一株高表达TRYR的主要乳杆菌菌株进行的双向凝胶分析显示，在TRYR的预测分子量上增加了5个斑点，在等电点等电点上相差0.08个pH单位。质谱学鉴定出其中四个是TRYR，这导致了新的建议，即它可能被翻译后修饰。最后，对体外产生的耐甲氨蝶呤突变体进行了定量比较分析，发现已知的主要抗性介体--蝶啶还原酶PTR1--过表达。这构成了对寄生虫耐药性的第一次蛋白质组学分析，也是使用这种方法对主要耐药性机制进行的最清晰的识别。总之，这些结果为利什曼原虫的进一步蛋白质组学研究提供了一个框架，并表明这些工具对于潜在的药物靶点和耐药机制的基本研究是有价值的。
Leishmania is a protozoan parasite responsible for significant morbidity and mortality worldwide. Few parasites have been subjected to proteomic analysis to date, but a genome sequencing project for Leishmania, major is currently underway, making these studies possible. Here we present a high resolution proteome for L. major comprising almost 3700 spots, making it the most complete two-dimensional gel representation of a parasite proteome generated to date. We have identified a number of landmark proteins by mass spectrometry and show that several of these are valid for the related species Leishmania donovani infantum. We have also observed several forms and fragments of alpha- and beta-tubulins and show that the number and amount of these fragments increase with the age of the parasite culture. Trypanothione reductase (TRYR), which replaces glutathione reductase in trypanosomatid parasites, is an essential protein specific to these parasites and as such is under considerable scrutiny as a drug target. Two-dimensional gel analysis of a L. major strain overexpressing TRYR revealed increased amounts of five spots, all at the predicted molecular weight for TRYR and differing by 0.08 pH units in pI. Mass spectrometry identified four of these as TRYR, leading to the novel suggestion that it could be posttranslationally modified. Finally quantitative comparative analysis of a methotrexate-resistant mutant of L. major generated in vitro found that a known primary resistance mediator, the pteridine reductase PTR1, was overexpressed. This constitutes the first proteomic analysis of drug resistance in a parasite and also the clearest identification of a primary drug resistance mechanism using this approach. Together these results provide a framework for further proteomic studies of Leishmania species and demonstrate that these tools are valuable for the essential study of potential drug targets and drug resistance mechanisms.