title: 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 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.
authors.affiliation: Departamento de Parasitologia, Instituto de Ciências Biomédicas, Universidade de São Paulo, São Paulo, Brazil.
authors.forename: Lisvane Silva
authors.initials: LS
authors.lastname: Paes
authors.forename: Brian
authors.initials: B
authors.lastname: Suárez Mantilla
authors.forename: Flávia Menezes
authors.initials: FM
authors.lastname: Zimbres
authors.forename: Elisabeth Mieko Furusho
authors.initials: EM
authors.lastname: Pral
authors.forename: Patrícia
authors.initials: P
authors.lastname: Diogo de Melo
authors.forename: Erich B
authors.initials: EB
authors.lastname: Tahara
authors.forename: Alicia J
authors.initials: AJ
authors.lastname: Kowaltowski
authors.forename: Maria Carolina
authors.initials: MC
authors.lastname: Elias
authors.forename: Ariel Mariano
authors.initials: AM
authors.lastname: Silber
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D015536:Down-Regulation; D004579:Electron Transport; D005182:Flavin-Adenine Dinucleotide / Q000378:metabolism; D042541:Intracellular Space / Q000378:metabolism; D008018:Life Cycle Stages; D008969:Molecular Sequence Data; D018384:Oxidative Stress; D011392:Proline / Q000378:metabolism; D011394:Proline Oxidase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D021381:Protein Transport; D014349:Trypanosoma cruzi / Q000166:cytology / Q000201:enzymology / Q000235:genetics / Q000378:metabolism*
chemical_list: D005182:Flavin-Adenine Dinucleotide; D011392:Proline; D011394:Proline Oxidase
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