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

谷胱甘肽氧化还原对作为疟疾寄生虫恶性疟原虫中硫醇开关的操纵者

• 批准号: 251796707
• 负责人: Dr. Stefan Rahlfs, since 1/2020
• 金额: --
• 依托单位: Professur für Biochemie und Molekularbiologie
• 依托单位国家: 德国
• 项目类别: Priority Programmes
• 财政年份: 2014
• 资助国家: 德国
• 起止时间: 2013-12-31 至 2021-12-31
• 项目状态: 已结题
• 来源: https://gepris.dfg.de/gepris/projekt/251796707?language=en
• 关键词: glutathione; redox; couple; thiol; switch

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.

由于其在细胞快速生长和繁殖中的重要功能，包括疟原虫在内的许多原生动物寄生虫的氧化还原代谢被认为是化疗预防和干预的主要目标。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，例如缺乏真正的过氧化氢酶和谷胱甘肽过氧化物酶，以及存在plasmoredoxin和四种不同的硒蛋白。此外，氧化还原信号是寄生虫-宿主细胞单位的一个主要调控原则，它参与疟疾的病理生理，并且一些抗疟疾药物至少部分通过基于氧化还原的机制调节其作用。在过去的几年里，我们深入研究了疟疾寄生虫中氧化还原相关酶的结构、功能、定位、相互作用和（谷胱甘肽依赖）氧化还原调节。在这里提出的SPP 1710项目中，我们的目标是通过关注基于硫醇开关的氧化还原传感和基于硫醇开关的蛋白质相互作用，进一步阐明恶性疟原虫中心氧化还原开关的功能和调控。基于硫醇开关的恶性疟原虫氧化还原感应。在第一个重点中，我们的目标是基因组整合和系统比较氧化还原探针hGrx1roGFP2和sfroGFP2，以及H2O2探针roGFP2Orp1和目前正在开发的新型H2O2探针。这将在寄生虫的细胞质和细胞器中产生强大的检测系统。只要可能，读数应从共聚焦激光扫描显微镜转移到平板阅读器。基因编码的氧化还原探针可用于研究疟疾寄生虫的药物作用机制和耐药性。我们进一步计划确定非应激和应激寄生虫的亚砜化水平。然后，通过SILAC方法，阐明氧化应激下谷胱甘肽酰化和亚砜酰化的定量和定性变化。与第一个资助期一样，将研究氧化还原调控硫醇修饰对所选酶的结构和功能的影响。基于巯基开关的蛋白质相互作用。在第二个重点研究中，我们计划使用PfPrx1a和PfPrx1m突变体的混合二硫钓鱼方法来鉴定这些过氧化物还毒素的氧化还原相互作用组。选择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种Prx及其氧化还原素相互作用中的作用将通过表面等离子体共振（SPR）进行研究。SPR在分析氧化还原相互作用和修饰方面的适用性应与ITC和微尺度热泳术进行系统比较。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。