Leopoldo de Meis Institute of Medical Biochemistry, Federal University of Rio de Janeiro, 21941-902, Rio de Janeiro, Brazil; Carlos Chagas Filho Institute of Biophysics,Federal University of Rio de Janeiro, 21941-902, Rio de Janeiro, Brazil; National Center of Structural Biology and Bioimaging, Federal University of Rio de Janeiro, 21941-902, Rio de Janeiro, Brazil.
Leopoldo de Meis Institute of Medical Biochemistry, Federal University of Rio de Janeiro, 21941-902, Rio de Janeiro, Brazil; National Institute of Science and Technology for Structural Biology, Federal University of Rio de Janeiro, 21941-902, Rio de Janeiro, Brazil.
Exp Parasitol. 2020 Oct;217:107962. doi: 10.1016/j.exppara.2020.107962. Epub 2020 Aug 4.
Trypanosoma cruzi is a parasitic protozoan that infects various species of domestic and wild animals, triatomine bugs and humans. It is the etiological agent of American trypanosomiasis, also known as Chagas Disease, which affects about 17 million people in Latin America and is emerging elsewhere in the world. Iron (Fe) is a crucial micronutrient for almost all cells, acting as a cofactor for several metabolic enzymes. T. cruzi has a high requirement for Fe, using heminic and non-heminic Fe for growth and differentiation. Fe occurs in the oxidized (Fe) form in aerobic environments and needs to be reduced to Fe before it enters cells. Fe-reductase, located in the plasma membranes of some organisms, catalyzes the Fe⇒ Fe conversion. In the present study we found an amino acid sequence in silico that allowed us to identify a novel 35 kDa protein in T. cruzi with two transmembrane domains in the C-terminal region containing His residues that are conserved in the Ferric Reductase Domain Superfamily and are required for catalyzing Fe reduction. Accordingly, we named this protein TcFR. Intact epimastigotes from the T. cruzi DM28c strain reduced the artificial Fe-containing substrate potassium ferricyanide in a cell density-dependent manner, following Michaelis-Menten kinetics. The TcFR activity was more than eightfold higher in a plasma membrane-enriched fraction than in whole homogenates, and this increase was consistent with the intensity of the 35 kDa band on Western blotting images obtained using anti-NOX5 raised against the human antigen. Immunofluorescence experiments demonstrated TcFR on the parasite surface. That TcFR is part of a catalytic complex allowing T. cruzi to take up Fe from the medium was confirmed by experiments in which DM28c was assayed after culturing in Fe-depleted medium: (i) proliferation during the stationary growth phase was five times slower; (ii) the relative expression of TcFR (qPCR) was 50% greater; (iii) intact cells had 120% higher Fe-reductase activity. This ensemble of results indicates that TcFR is a conserved enzyme in T. cruzi, and its catalytic properties are modulated in order to respond to external Fe fluctuations.
克氏锥虫是一种寄生原生动物，感染各种家养和野生动物、三锥虫和人类。它是美洲锥虫病（也称为恰加斯病）的病原体，这种疾病影响拉丁美洲约 1700 万人，并且正在世界其他地方出现。铁（Fe）是几乎所有细胞的重要微量营养素，作为几种代谢酶的辅助因子。克氏锥虫对 Fe 的需求量很高，它使用血红素和非血红素 Fe 来生长和分化。在有氧环境中，Fe 以氧化（Fe）形式存在，需要还原为 Fe 才能进入细胞。Fe 还原酶位于一些生物体的质膜中，催化 Fe ⇒ Fe 的转化。在本研究中，我们在计算机中发现了一个氨基酸序列，使我们能够在克氏锥虫中鉴定出一种新型的 35 kDa 蛋白，该蛋白在 C 端区域含有两个跨膜结构域，其中含有保守的亚铁还原酶结构域超家族残基，这些残基对于催化 Fe 还原是必需的。因此，我们将这种蛋白命名为 TcFR。来自 T. cruzi DM28c 株的完整的epimastigotes 以细胞密度依赖的方式还原人工含 Fe 的底物铁氰化钾，遵循米氏动力学。与全匀浆相比，TcFR 活性在富含质膜的级分中高出 8 倍以上，并且这种增加与使用针对人抗原 NOX5 产生的抗体制备的 Western blot 图像中 35 kDa 带的强度一致。免疫荧光实验证明 TcFR 在寄生虫表面。通过在缺铁培养基中培养 DM28c 后进行的实验证实 TcFR 是一种催化复合物的一部分，该复合物允许 T. cruzi 从培养基中摄取 Fe：（i）静止生长阶段的增殖速度慢了五倍；（ii）TcFR（qPCR）的相对表达增加了 50%；（iii）完整细胞的铁还原酶活性增加了 120%。这一系列结果表明 TcFR 是克氏锥虫中的一种保守酶，其催化特性受到调节以响应外部 Fe 波动。
