Trypanosoma cruzi synthesizes proline via a Δ1-pyrroline-5-carboxylate reductase whose activity is fine-tuned by NADPH cytosolic pools

Abstract

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.

Developmental changes and metabolic reprogramming during establishment of infection and progression of Trypanosoma brucei brucei through its insect host

“…The former has FBPase-2 activity, which converts fructose 2,6-bisphosphate to fructose-6-phosphate. Although proventricular forms express their own subset of proline/alanine transporters it is possible that they are synthesising proline as well, since cytoplasmic delta-1-pyrroline-5-carboxylate reductase is upregulated [ 77 ]. The complexity of the population in the salivary glands does not allow us to attribute metabolic pathways to specific life-cycle stages.…”

Developmental changes and metabolic reprogramming during establishment of infection and progression of Trypanosoma brucei brucei through its insect host

“…This suggests that the protein compositions of the glycosome and the mitochondrion change over time as the trypanosome moves from a reliance on glycolysis to proline catabolism. Although proventricular forms express their own subset of proline/alanine transporters it is possible that they are synthesising proline as well, since cytoplasmic delta-1-pyrroline-5carboxylate reductase is upregulated (74). Proventricular forms also express two isoforms of PFK-2/FBPase-2, which can catalyse both glycolysis and gluconeogenesis.…”

Mitochondrial adaptations throughout the Trypanosoma brucei life cycle

“…Electrons are supplied either directly to ubiquinone by the membrane‐bound proline dehydrogenase or indirectly via reduced NADH molecules generated by pyrroline‐5‐carboxylate dehydrogenase. In the latter scenario, the NADH is subsequently oxidized by complex I and/or NDH2 (Bringaud et al, 2012; Marchese et al, 2020).…”