Genetically encoded biosensors for monitoring redox changes in the trypanothione-based thiol metabolism of trypanosomes

锥虫和利什曼原虫缺乏谷胱甘肽还原酶和硫氧还蛋白还原酶，这两种酶几乎无处不在，负责维持细胞内还原环境。寄生原生动物的硫醇氧化还原动态平衡是以色硫酮为基础的，色硫酮是由多胺亚精胺和两个谷胱甘肽分子组成的偶联物。所有参与台盼硫酮生物合成和还原的酶都位于细胞的胞浆中。在这里，通过硫醇/二硫键交换，台盼硫可还原谷胱甘肽二硫化物以及氧化还原蛋白，如色氨酸、硫氧还蛋白和谷氧还蛋白。对总谷胱甘肽、谷胱甘肽亚精胺和色氨硫酮的测量表明，当引起非洲昏睡病和Nagana牛疾病的血液形式的布氏锥虫暴露于外源性和内源性氧化应激时，细胞低分子硫醇库发生了非常明显的变化。然而，这种类型的分析不能提供关于不同细胞室中具体情况的任何信息。该项目的目的是建立在布氏毛滴虫单个线粒体的胞浆、膜间隙和基质中表达各种氧化还原活性绿色荧光蛋白(RoGFP)的细胞系，从而能够对完整寄生虫在稳态和硫醇开关条件下的硫醇氧化还原状态进行特定的分析。将解决的主要问题是：整个细胞的硫醇状态是否反映了胞浆中的条件？胞浆和线粒体的硫醇氧化还原代谢之间是否存在相互作用？与感染性血液寄生虫的基本细胞器中的硫醇氧化还原状态相比，昆虫阶段充分展开的线粒体中的硫醇氧化还原状态如何？台盼硫酮生物合成或还原的下调如何影响胞质和线粒体的硫醇氧化还原状态？这两种谷氧还蛋白分别定位于胞浆和线粒体膜间间隙，是否起到硫醇氧化还原开关的作用？计划中的工作应该会对非洲锥虫的胞浆和线粒体硫醇代谢有深刻的了解。此外，稳定的细胞系有望成为未来研究寄生虫氧化还原调节过程(如分化)的诱人工具。

Trypanosomes and Leishmania lack both glutathione reductases and thioredoxin reductase, nearly ubiquitous enzymes that are responsible for maintaining the reducing intracellular milieu. The thiol redox homeostasis of the parasitic protozoa is based on trypanothione, a conjugate composed of the polyamine spermidine and two molecules of glutathione. All enzymes involved in the biosynthesis and reduction of trypanothione are located in the cytosol of the cell. Here, by thiol/disulfide exchange, trypanothione reduces glutathione disulfide as well as redox proteins such tryparedoxin, thioredoxin and glutaredoxins. Measurements of total glutathione, glutathionylspermidine and trypanothione revealed that the cellular low molecular mass thiol pool undergoes highly distinct changes when the bloodstream form of Trypanosoma brucei , the causative agent of African sleeping sickness and Nagana cattle disease, is exposed to exogenous and endogenous oxidative stresses. This type of analysis, however, cannot provide any information about the specific situation in the different cell compartments.Aim of this project is to generate cell lines that express various redox active green fluorescent proteins (roGFPs) in the cytosol as well as intermembrane space and matrix of the single mitochondrion of T. brucei and thus allow a compartment-specific analysis of the thiol redox status under steady state and thiol switch conditions in the intact parasites. The main questions that will be addressed are: Does the whole cell thiol status reflect the conditions in the cytosol? Is there a cross-talk between the thiol redox metabolism of the cytosol and the mitochondrial compartments? How does the thiol redox status in the fully elaborated mitochondrion of the insect stage compare with that in the rudimentary organelle of the infectious bloodstream parasite? How does down-regulation of the trypanothione biosynthesis or reduction affect the cytosolic and mitochondrial thiol redox status? Do the two glutaredoxins, localized in the cytosol and mitochondrial intermembrane space, respectively, act as thiol redox switches?The planned work should give a profound insight into the cytosolic and mitochondrial thiol metabolism of African trypanosomes. In addition, the stable cell lines generated are expected to be attractive tools for future studies of putatively redox regulated processes in the parasites such as differentiation.