Marchese Letícia, Olavarria Karel, Mantilla Brian Suarez, Avila Carla Cristi, Souza Rodolpho Ornitiz Oliveira, Damasceno Flávia Silva, Elias Maria Carolina, Silber Ariel Mariano
Laboratory of Biochemistry of Tryps - LaBTryps, Department of Parasitology, Institute of Biomedical Sciences, University of São Paulo - USP, 05508-000 São Paulo, Brazil.
Department of Microbiology, Institute of Biomedical Sciences, University of São Paulo - USP, 05508-000 São Paulo, Brazil.
Biochem J. 2020 May 29;477(10):1827-1845. doi: 10.1042/BCJ20200232.
In Trypanosoma cruzi, the etiological agent of Chagas disease, the amino acid proline participates in processes related to T. cruzi survival and infection, such as ATP production, cell differentiation, host-cell invasion, and in protection against osmotic, nutritional, and thermal stresses and oxidative imbalance. However, little is known about proline biosynthesis in this parasite. Δ1-Pyrroline-5-carboxylate reductase (P5CR, EC 1.5.1.2) catalyzes the biosynthesis of proline from Δ1-pyrroline-5-carboxylate (P5C) with concomitant NADPH oxidation. Herein, we show that unlike other eukaryotes, T. cruzi biosynthesizes proline from P5C, which is produced exclusively from glutamate. We found that TcP5CR is an NADPH-dependent cytosolic enzyme with a Kmapp for P5C of 27.7 μM and with a higher expression in the insect-resident form of the parasite. High concentrations of the co-substrate NADPH partially inhibited TcP5CR activity, prompting us to analyze multiple kinetic inhibition models. The model that best explained the obtained data included a non-competitive substrate inhibition mechanism (Kiapp=45±0.7μM). Therefore, TcP5CR is a candidate as a regulatory factor of this pathway. Finally, we show that P5C can exit trypanosomatid mitochondria in conditions that do not compromise organelle integrity. These observations, together with previously reported results, lead us to propose that in T. cruzi TcP5CR participates in a redox shuttle between the mitochondria and the cytoplasm. In this model, cytoplasmic redox equivalents from NADPH pools are transferred to the mitochondria using proline as a reduced metabolite, and shuttling to fuel electrons to the respiratory chain through proline oxidation by its cognate dehydrogenase.
在恰加斯病的病原体克氏锥虫中，氨基酸脯氨酸参与了与克氏锥虫存活和感染相关的过程，如三磷酸腺苷（ATP）生成、细胞分化、宿主细胞入侵，以及抵御渗透压、营养和热应激及氧化失衡。然而，对于这种寄生虫中脯氨酸的生物合成知之甚少。Δ1-吡咯啉-5-羧酸还原酶（P5CR，EC 1.5.1.2）催化从Δ1-吡咯啉-5-羧酸（P5C）生物合成脯氨酸，并伴随烟酰胺腺嘌呤二核苷酸磷酸（NADPH）氧化。在此，我们表明，与其他真核生物不同，克氏锥虫从P5C生物合成脯氨酸，而P5C仅由谷氨酸产生。我们发现，克氏锥虫P5CR是一种依赖NADPH的胞质酶，对P5C的表观米氏常数（Kmapp）为27.7 μM，并且在寄生虫的昆虫寄生形式中表达较高。高浓度的共底物NADPH部分抑制了克氏锥虫P5CR的活性，促使我们分析多种动力学抑制模型。最能解释所得数据的模型包括非竞争性底物抑制机制（表观抑制常数Kiapp = 45±0.7 μM）。因此，克氏锥虫P5CR是该途径调控因子的一个候选者。最后，我们表明，在不损害细胞器完整性的条件下，P5C可以从锥虫线粒体中输出。这些观察结果，连同先前报道的结果，使我们提出，在克氏锥虫中，克氏锥虫P5CR参与线粒体与细胞质之间的氧化还原穿梭。在这个模型中，来自NADPH池的细胞质氧化还原当量以脯氨酸作为还原代谢物转移到线粒体，并通过其同源脱氢酶对脯氨酸的氧化穿梭为呼吸链提供电子。
