Proline Dehydrogenase Regulates Redox State and Respiratory Metabolism in Trypanosoma cruzi

Abstract

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 Δ1-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.

Role of Δ1-Pyrroline-5-Carboxylate Dehydrogenase Supports Mitochondrial Metabolism and Host-Cell Invasion of Trypanosoma cruzi

“…The first enzymatic step of this pathway consists of the conversion of proline into P5C, as has been recently elucidated (10). In this study, we provide conclusive biochemical, mechanistic, and molecular evidence of the functionality of TcP5CDH in several aspects of the T. cruzi life cycle.…”

“…This relationship has also been confirmed in several pathogenic trypanosomatids (10,31). However, there is no evidence to indicate a role for P5CDH in mitochondrial functions.…”

“…First, proline is converted to ⌬ 1 -pyrroline-5-carboxylate (P5C) 2 through a FAD (cofactor)-dependent reaction that is catalyzed by a proline dehydrogenase (TcProDH) (EC 1.5.99.8) (10). The subsequent step involves a nonenzymatic reaction in which the cyclic P5C ring is spontaneously opened, producing glutamate-␥-semialdehyde (␥GS) (18).…”

Caracterização molecular e funcional da imidazolona propionase de <i>Trypanosoma cruzi</i>: uma enzima do metabolismo de histidina.

“…Sabe-se que a Pro é degradada em dois passos enzimáticos a Glu: primeiro, é oxidada em Δ1-pirrolina-5-carboxilato (P5C), reação catalisada pela prolina desidrogenase (TcPRODH) e posteriormente o mesmo é convertido em Glu pela Δ1-pirrolina-5-carboxilato desidrogenase (TcP5CDH) na presença de NAD(P) + . Foi descrito que a oxidação de Pro acontece na mitocôndria, estimulando a respiração celular e sustentando a síntese de ATP através do processo de fosforilação oxidativa (Paes et al, 2013;Mantilla et al, 2015).…”

“…Foi observado que além de incorporada pelo parasita, His está sendo usada como fonte de energia para manter a viabilidade celular (Barison et al, 2016), em níveis similares aos demonstrados para Pro, anteriormente descrita como um aminoácido protetor ao estresse metabólico e relevante em termos de bioenergética para o parasita (Magdaleno et al, 2009;Martins et al, 2009;Paes et al, 2013;Mantilla et al, 2015).…”