An essential thioredoxin-type protein of Trypanosoma brucei acts as redox-regulated mitochondrial chaperone
An essential thioredoxin-type protein of Trypanosoma brucei acts as redox-regulated mitochondrial chaperone
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DOI：
10.1371/journal.ppat.1008065
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发表时间：
2019-09-01
期刊：
影响因子：
6.7
通讯作者：
Krauth-Siegel, R. Luise
中科院分区：
文献类型：
--
作者：
Currier, Rachel B.;Ulrich, Kathrin;Krauth-Siegel, R. Luise
大多数已知的硫氧还蛋白类蛋白(TRX)参与氧化还原途径，使用两个高度保守的半胱氨酸残基催化硫醇-二硫键交换反应。在这里，我们证明了到目前为止还没有发现的来自非洲锥虫(Trypanosoma Brucei)的Trx2缺乏蛋白质二硫键还原酶活性，但作为一种有效的温度激活和氧化还原调节的伴侣功能。免疫荧光显微镜和分级细胞裂解显示Trx2位于寄生虫的线粒体中。RNA干扰和基因敲除方法表明，Trx2的缺失会损害哺乳动物血流和昆虫阶段原环寄生虫的生长。缺少Trx2的原周期细胞在27摄氏度的标准培养条件下停止增殖，并无法长期暴露在37摄氏度下，这表明Trx2在热应激下发挥着至关重要的作用。此外，我们还发现Trx2与布氏毛滴虫的体内感染性有关。值得注意的是，Trx2版本，其中所有五个半胱氨酸都被丝氨酸残基取代，补充了条件基因敲除细胞中的野生型蛋白，并在小鼠模型中赋予了寄生虫感染性。对重组蛋白的鉴定表明，Trx2能配位铁硫簇，对自发氧化高度敏感。此外，我们发现野生型和突变型Trx2都保护其他蛋白质免受热聚集，并保持它们在返回非应激条件下重新折叠的能力。Trx2的伴侣功能的激活似乎是由温度介导的结构变化触发的，并被氧化二硫键的形成抑制。我们的研究表明，Trx2是布鲁氏锥虫独特的单个线粒体中的一种新的伴侣蛋白，为锥虫硫氧还蛋白类蛋白的生理功能提供了一个新的视角。这些严格的细胞外病原体在它们的哺乳动物宿主和采采蝇载体的血液和体液中繁殖，在那里有效的氧化还原调节对寄生虫的生存至关重要。虽然大多数生物使用谷胱甘肽/谷胱甘肽还原酶和硫氧还蛋白/硫氧还蛋白还原酶对来维持细胞的氧化还原平衡，但锥虫依赖于一种独特的基于色氨硫酮的硫醇代谢来生存外源和内源氧化应激。尽管缺乏硫氧还蛋白还原酶，但布鲁氏锥虫基因组编码硫氧还蛋白，这引发了人们对其生物学功能的质疑。我们的工作是首次报道布氏毛滴虫硫氧还蛋白-2(Trx2)。我们发现Trx2位于线粒体中，它的缺失会影响寄生虫的增殖和感染性。重组Trx2缺乏蛋白质二硫键还原酶活性，但保护蛋白质不聚集并保持其折叠能力。值得注意的是，在体外和体内条件下，没有任何半胱氨酸残基的突变体能够完全取代真正的蛋白质。我们的数据显示，Trx2并不是一个经典的硫氧还蛋白，而是一个在布鲁氏毛滴虫线粒体中起关键作用的伴侣。
Most known thioredoxin-type proteins (Trx) participate in redox pathways, using two highly conserved cysteine residues to catalyze thiol-disulfide exchange reactions. Here we demonstrate that the so far unexplored Trx2 from African trypanosomes (Trypanosoma brucei) lacks protein disulfide reductase activity but functions as an effective temperature-activated and redox-regulated chaperone. Immunofluorescence microscopy and fractionated cell lysis revealed that Trx2 is located in the mitochondrion of the parasite. RNA-interference and gene knock-out approaches showed that depletion of Trx2 impairs growth of both mammalian bloodstream and insect stage procyclic parasites. Procyclic cells lacking Trx2 stop proliferation under standard culture conditions at 27 degrees C and are unable to survive prolonged exposure to 37 degrees C, indicating that Trx2 plays a vital role that becomes augmented under heat stress. Moreover, we found that Trx2 contributes to the in vivo infectivity of T. brucei. Remarkably, a Trx2 version, in which all five cysteines were replaced by serine residues, complements for the wildtype protein in conditional knock-out cells and confers parasite infectivity in the mouse model. Characterization of the recombinant protein revealed that Trx2 can coordinate an iron sulfur cluster and is highly sensitive towards spontaneous oxidation. Moreover, we discovered that both wildtype and mutant Trx2 protect other proteins against thermal aggregation and preserve their ability to refold upon return to non-stress conditions. Activation of the chaperone function of Trx2 appears to be triggered by temperature-mediated structural changes and inhibited by oxidative disulfide bond formation. Our studies indicate that Trx2 acts as a novel chaperone in the unique single mitochondrion of T. brucei and reveal a new perspective regarding the physiological function of thioredoxin-type proteins in trypanosomes.Author summary African trypanosomes are the causative agents of human sleeping sickness and Nagana cattle disease. These strictly extracellular pathogens multiply in the blood and body fluids of their mammalian hosts and the tsetse fly vector, where efficient redox regulation is essential for parasite survival. While most organisms use the glutathione/glutathione reductase and thioredoxin/thioredoxin reductase couples to maintain cellular redox balance, trypanosomes rely on a unique trypanothione-based thiol metabolism to survive exogenous and endogenous oxidative stresses. Despite the lack of thioredoxin reductases, the Trypanosoma brucei genome encodes thioredoxins, raising questions for their biological function. Our work is the first report on T. brucei thioredoxin-2 (Trx2). We show that Trx2 is located in the mitochondrion and its absence affects parasite proliferation and infectivity. Recombinant Trx2 lacks protein disulfide reductase activity but protects proteins against aggregation and maintains them folding-competent. Remarkably, a mutant that is devoid of any cysteine residues is able to fully substitute for the authentic protein under in vitro and in vivo conditions. Our data reveal that Trx2 does not function as a classical thioredoxin but acts as a chaperone that plays a crucial role in the mitochondrion of T. brucei.