The glutathione redox couple as a thiol switch operator in the malaria parasite Plasmodium falciparum

由于其在细胞快速生长和繁殖中的重要作用，包括疟疾寄生虫在内的许多原虫的氧化还原代谢被认为是化疗预防和干预的主要靶点。值得注意的是，疟疾寄生虫中的氧化还原网络具有独特的特征，例如不存在真正的过氧化氢酶和谷胱甘肽过氧化物酶，而存在浆还蛋白和四种不同的硒蛋白。此外，氧化还原信号是寄生虫-宿主细胞单位的主要调控原理，参与了疟疾的病理生理过程，几种抗疟药物至少部分地通过氧化还原机制介导了它们的作用。在过去的几年里，我们在结构、功能、定位、相互作用和(谷胱甘肽依赖的)氧化还原调节方面对疟疾寄生虫的氧化还原相关酶进行了深入的研究。在这里提出的SPP1710项目中，我们的目标是通过基于硫醇开关的氧化还原感应和基于硫醇开关的蛋白质相互作用来进一步阐明恶性疟原虫中心氧化还原开关的功能和调节。恶性疟原虫中基于硫醇开关的氧化还原感应。在第一个焦点中，我们的目标是对氧化还原探针hGrx1roGFP2和sFroGFP2以及过氧化氢探针roGFP2Orp1和目前正在开发的新型过氧化氢探针进行基因组整合和系统比较。这将在寄生虫的细胞质和细胞器中产生强大的检测系统。只要有可能，读数应从共聚焦激光扫描显微镜转移到平板阅读器上。应使用基因编码的氧化还原探针来研究疟疾寄生虫的药物作用机制和耐药性。我们进一步计划测定非应激和应激寄生虫的硫苯化水平。然后，在SILAC方法中，应阐明氧化应激下谷胱甘肽基化和直接比较中亚硫苯基化的数量和质量变化。与第一个资助期一样，应研究氧化还原调节硫醇修饰对选定酶的结构和功能的影响。基于硫醇开关的蛋白质相互作用。在第二个焦点中，使用PfPrx1a和PfPrx1m突变体的混合二硫键钓鱼方法，我们计划鉴定这些过氧化还蛋白的氧化还原相互作用体。选定的相互作用将使用重组蛋白进行验证。不同的半胱氨酸残基在两个Prx和它们的氧化还原蛋白相互作用中的作用将通过表面等离子体共振(SPR)来研究。SPR在分析氧化还原相互作用和修饰方面的适用性应与ITC和微尺度热电泳法进行系统比较。与SPP联盟的其他小组合作，SPR将用于研究蛋白质相互作用、靶标特异性和反应性。

Due to its crucial functions in rapidly growing and multiplying cells, the redox metabolism of many protozoan parasites including the malaria parasite Plasmodium is considered as a major target for chemotherapeutic prophylaxis and intervention. Notably, redox networks in malaria parasites have unique features such as the absence of a genuine catalase and glutathione peroxidase and the presence of plasmoredoxin and four distinct selenoproteins. Furthermore, redox signaling is a major regulatory principle in the parasite-host cell unit, it is involved in the pathophysiology of malaria, and several antimalarial drugs mediate their effects at least partially by redox-based mechanisms. Over the last years we have intensely studied redox-related enzymes in malaria parasites with respect to structure, function, localization, interaction, and (glutathione dependent) redox regulation. Within the SPP 1710 project proposed here, we aim to further elucidate the function and regulation of central redox switches in P. falciparum by focusing on thiol switch-based redox sensing and thiol switch-based interactions of proteins.1. Thiol switch-based redox sensing in P. falciparum. Within this first focus, we aim for genomic integration and systematic comparison of the redox probes hGrx1roGFP2 and sfroGFP2 as well as the H2O2 probe roGFP2Orp1 and novel H2O2 probes currently being developed. This shall result in robust detection systems in cytosol and organelles of the parasite. Wherever possible, the readout shall be transferred from the confocal laser scanning microscope to a plate reader. The genetically encoded redox probes shall be employed to study mechanisms of drug action and drug resistance in malaria parasites. We further plan to determine sulfenylation levels in unstressed and stressed parasites. Then, in a SILAC approach, the quantitative and qualitative changes in glutathionylation and, in direct comparison, sulfenylation under oxidative stress shall be elucidated. As in the first funding period, the impact of redox-regulatory thiol modifications on structure and function of selected enzymes shall be studied.2. Thiol switch-based interaction of proteins. In this second focus, using a mixed-disulfide fishing approach with mutants of PfPrx1a and PfPrx1m, we plan to identify the redox interactome of these peroxiredoxins. Selected interactions will be verified using recombinant proteins. The role of distinct cysteine residues in the interaction between the two Prx and their redoxins will be studied by surface plasmon resonance (SPR). The suitability of SPR in analyzing redox interactions and modifications shall be systematically compared with ITC and microscale thermophoresis. In cooperation with other groups of the SPP consortium, SPR will be employed to study protein interactions, target specificity and reactivity.