Departamento de Parasitologia, Instituto de Ciências Biomédicas, Universidade de São Paulo, São Paulo, Brazil.
PLoS One. 2013 Jul 22;8(7):e69419. doi: 10.1371/journal.pone.0069419. Print 2013.
Over the past three decades, L-proline has become recognized as an important metabolite for trypanosomatids. It is involved in a number of key processes, including energy metabolism, resistance to oxidative and nutritional stress and osmoregulation. In addition, this amino acid supports critical parasite life cycle processes by acting as an energy source, thus enabling host-cell invasion by the parasite and subsequent parasite differentiation. In this paper, we demonstrate that L-proline is oxidized to Δ(1)-pyrroline-5-carboxylate (P5C) by the enzyme proline dehydrogenase (TcPRODH, E.C. 1.5.99.8) localized in Trypanosoma cruzi mitochondria. When expressed in its active form in Escherichia coli, TcPRODH exhibits a Km of 16.58±1.69 µM and a Vmax of 66±2 nmol/min mg. Furthermore, we demonstrate that TcPRODH is a FAD-dependent dimeric state protein. TcPRODH mRNA and protein expression are strongly upregulated in the intracellular epimastigote, a stage which requires an external supply of proline. In addition, when Saccharomyces cerevisiae null mutants for this gene (PUT1) were complemented with the TcPRODH gene, diminished free intracellular proline levels and an enhanced sensitivity to oxidative stress in comparison to the null mutant were observed, supporting the hypothesis that free proline accumulation constitutes a defense against oxidative imbalance. Finally, we show that proline oxidation increases cytochrome c oxidase activity in mitochondrial vesicles. Overall, these results demonstrate that TcPRODH is involved in proline-dependant cytoprotection during periods of oxidative imbalance and also shed light on the participation of proline in energy metabolism, which drives critical processes of the T. cruzi life cycle.
在过去的三十年中，L-脯氨酸已被确认为动质体生物的一种重要代谢物。它参与了许多关键过程，包括能量代谢、对氧化和营养应激的抗性以及渗透压调节。此外，这种氨基酸通过充当能量来源来支持寄生虫关键生命周期过程，从而使寄生虫能够入侵宿主细胞并随后进行寄生虫分化。在本文中，我们证明 L-脯氨酸被定位在克氏锥虫线粒体中的酶脯氨酸脱氢酶（TcPRODH，E.C.1.5.99.8）氧化为 Δ(1)-吡咯啉-5-羧酸（P5C）。当在其活性形式在大肠杆菌中表达时，TcPRODH 的 Km 值为 16.58±1.69 µM，Vmax 值为 66±2 nmol/min mg。此外，我们证明 TcPRODH 是一种 FAD 依赖性二聚体蛋白。在需要外部脯氨酸供应的细胞内无鞭毛体阶段，TcPRODH mRNA 和蛋白表达强烈上调。此外，当该基因（PUT1）的酿酒酵母 null 突变体用 TcPRODH 基因互补时，与 null 突变体相比，观察到细胞内游离脯氨酸水平降低和对氧化应激的敏感性增强，这支持了游离脯氨酸积累构成对抗氧化失衡的防御假说。最后，我们表明脯氨酸氧化增加了线粒体囊泡中的细胞色素 c 氧化酶活性。总之，这些结果表明 TcPRODH 参与了氧化失衡期间依赖脯氨酸的细胞保护，还揭示了脯氨酸在能量代谢中的参与，这推动了克氏锥虫生命周期的关键过程。
