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

2020 ◽  
Vol 477 (10) ◽  
pp. 1827-1845
Author(s):  
Letícia Marchese ◽  
Karel Olavarria ◽  
Brian Suarez Mantilla ◽  
Carla Cristi Avila ◽  
Rodolpho Ornitiz Oliveira Souza ◽  
...  
Keyword(s):  
Trypanosoma Cruzi ◽  
Thermal Stresses ◽  
Substrate Inhibition ◽  
Inhibition Mechanism ◽  
Atp Production ◽  
Kinetic Inhibition ◽  
Redox Shuttle ◽  
High Concentrations ◽  
Competitive Substrate ◽  
Carboxylate Reductase

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.

scholarly journals Rationally Design of a Proline Transporter Inhibitor with Anti-Trypanosoma Cruzi Activity

2019 ◽  
Author(s):  
Lucia Fargnoli ◽  
Esteban A. Panozzo-Zénere ◽  
Lucas Pagura ◽  
María Julia Barisón ◽  
Julia A. Cricco ◽  
...  
Keyword(s):  
Amino Acid ◽  
Trypanosoma Cruzi ◽  
Chagas Disease ◽  
Variable Region ◽  
Amino Acid Uptake ◽  
Acid Uptake ◽  
Cell Infection ◽  
Atp Production ◽  
First Case

L-Proline is an important amino acid for the pathogenic protists belonging to <i>Trypanosoma</i> and <i>Leishmania </i>genera. In <i>Trypanosoma cruzi</i>, the etiological agent of Chagas disease, this amino acid is involved in fundamental biological processes such as ATP production, differentiation of the insect and intracellular stages, the host cell infection and the resistance to a variety of stresses, including nutritional and osmotic as well as oxidative imbalance. In this study, we explore the L-Proline uptake as a chemotherapeutic target for <i>T. cruzi</i>. For this, we propose a novel rational to design inhibitors containing this amino acid as a recognizable motif. This rational consists of conjugating the amino acid (proline in this case) to a linker and a variable region able to block the transporter. We obtained a series of sixteen 1,2,3-triazolyl-proline derivatives through alkylation and copper(I)-catalyzed azide-alkyne cycloaddition (click chemistry) for <i>in vitro</i> screening against <i>T. cruzi </i>epimastigotes, trypanocidal activity and proline uptake. We successfully obtained inhibitors that are able to interfere with the amino acid uptake, which validated the first example of a rationally designed chemotherapeutic agent targeting a metabolite's transport. Additionally, we designed and prepared fluorescent analogues of the inhibitors that were successfully taken up by <i>T. cruzi</i>, allowing following up their intracellular fate. In conclusion, we successfully designed and produced a series of metabolite uptake inhibitors. This is one of few examples of rationally designed amino acid transporter inhibitor, being the first case where the strategy is applied on the development of chemotherapy against Chagas disease. This unprecedented development is remarkable having in mind that only a small percent of the metabolite transporters has been studied at the structural and/or molecular level.

Gamma-glutamyltransferase: Substrate inhibition, kinetic mechanism, and assay conditions.

1976 ◽  
Vol 22 (4) ◽  
pp. 417-421 ◽  
Author(s):  
J H Stromme ◽  
L Theodorsen
Keyword(s):  
Clinical Chemistry ◽  
Substrate Inhibition ◽  
Kinetic Mechanism ◽  
Reaction Conditions ◽  
Gamma Glutamyltransferase ◽  
Gamma Glutamyl ◽  
Dead End ◽  
Ping Pong ◽  
Competitive Substrate ◽  
Assay Conditions

Abstract Gamma-glutamyltransferase activity in serum is shown to be competitively inhibited by the two substrates gamma-glutamyl-4-nitroanilide and glycylglycine. Awareness of this is of importance when one is choosing final reaction conditions for the assay of the enzyme. Gamma-glutamyltransferase probably acts by a "ping-pong bi-bi" kinetic mechanism, which fits with the double competitive substrate inhibition demonstrated. The product, 4-nitro-aniline, appears to be an uncompetitive dead-end inhibitor of both substrates. Various amino acids, particularly glycine and L-alanine, inhibit the enzyme. Their inhibition patterns are uncompetitive with glycylglycine and competitive with gamma-glutamyl-4-nitroanilide. On the basis of the present and other studies, the Scandinavian Society for Clinical Chemistry and Clinical Physiology is going to recommend for routine use a gamma-glutamyltransferase method in which the final concentrations of gamma-glutamyl-4-nitroanilide and glycylglycine are 4 and 75 mmol/liter, respectively.

The type IV pilus assembly motor PilB is a robust hexameric ATPase with complex kinetics

2018 ◽  
Vol 475 (11) ◽  
pp. 1979-1993 ◽  
Author(s):  
Andreas Sukmana ◽  
Zhaomin Yang
Keyword(s):  
Atpase Activity ◽  
Enrichment Culture ◽  
Physiological State ◽  
Substrate Inhibition ◽  
Kinetic Studies ◽  
Enzyme Analysis ◽  
Type Iv Pilus ◽  
Type Iv ◽  
High Concentrations

The bacterial type IV pilus (T4P) is a versatile nanomachine that functions in pathogenesis, biofilm formation, motility, and horizontal gene transfer. T4P assembly is powered by the motor ATPase PilB which is proposed to hydrolyze ATP by a symmetrical rotary mechanism. This mechanism, which is deduced from the structure of PilB, is untested. Here, we report the first kinetic studies of the PilB ATPase, supporting co-ordination among the protomers of this hexameric enzyme. Analysis of the genome sequence of Chloracidobacterium thermophilum identified a pilB gene whose protein we then heterologously expressed. This PilB formed a hexamer in solution and exhibited highly robust ATPase activity. It displays complex steady-state kinetics with an incline followed by a decline over an ATP concentration range of physiological relevance. The incline is multiphasic and the decline signifies substrate inhibition. These observations suggest that variations in intracellular ATP concentrations may regulate T4P assembly and T4P-mediated functions in vivo in accordance with the physiological state of bacteria with unanticipated complexity. We also identified a mutant pilB gene in the genomic DNA of C. thermophilum from an enrichment culture. The mutant PilB variant, which is significantly less active, exhibited similar inhibition of its ATPase activity by high concentrations of ATP. Our findings here with the PilB ATPase from C. thermophilum provide the first line of biochemical evidence for the co-ordination among PilB protomers consistent with the symmetrical rotary model of catalysis based on structural studies.

Measurement of Isocitric Dehydrogenase Activity in Body Fluids

1959 ◽  
Vol 5 (6) ◽  
pp. 509-518 ◽  
Author(s):  
George N Bowers
Keyword(s):  
Standard Deviation ◽  
Dehydrogenase Activity ◽  
Substrate Inhibition ◽  
Body Fluids ◽  
Isocitric Dehydrogenase ◽  
Factors Influencing ◽  
Activity Curve ◽  
High Concentrations ◽  
Healthy Males

Abstract 1. Factors influencing the isocitric dehydrogenase (ICD) activity of serum were studied. Substrate inhibition by high concentrations of isocitrate was observed. 2. The temperature-activity curve of serum ICD was determined. The importance of controlling the temperature of the reaction was stressed. 3. Using this information, a method of measuring the ICD activity in 0.2 ml. of serum is described. 4. The range of ICD activity in 50 healthy males was 30 to 192 units with a mean of 90 units and a standard deviation of ± 34.3 units.

Competitive substrate inhibition of amyloglucosidase fromRhizopussp. by vanillin and curcumin

2006 ◽  
Vol 24 (4) ◽  
pp. 299-305 ◽  
Author(s):  
Ramaiah Sivakumar ◽  
Giriyapura R. Vijayakumar ◽  
Balaraman Manohar ◽  
Soundar Divakar
Keyword(s):  
Substrate Inhibition ◽  
Competitive Substrate

Computational biotechnology: Prediction of competitive substrate inhibition of enzymes by buffer compounds with protein–ligand docking

2012 ◽  
Vol 161 (4) ◽  
pp. 391-401 ◽  
Author(s):  
Karen T. Schomburg ◽  
Inés Ardao ◽  
Katharina Götz ◽  
Fabian Rieckenberg ◽  
Andreas Liese ◽  
...  
Keyword(s):  
Substrate Inhibition ◽  
Ligand Docking ◽  
Competitive Substrate

scholarly journals Higher expression of proline dehydrogenase altered mitochondrial function and increased Trypanosoma cruzi differentiation in vitro and in the insect vector

2021 ◽  
Author(s):  
Brian S Mantilla ◽  
Lisvane Paes-Vieira ◽  
Felipe Almeida Dias ◽  
Simone G. Caldeirano ◽  
Maria Carolina Elias ◽  
...  
Keyword(s):  
Trypanosoma Cruzi ◽  
Insect Vector ◽  
Mitochondrial Enzymes ◽  
Proline Dehydrogenase ◽  
Intermediate Hosts ◽  
Atp Production ◽  
Metacyclic Trypomastigotes ◽  
Transport Chain ◽  
Mitochondrial Functions

The pathogenic protist Trypanosoma cruzi uses kissing bugs as intermediate hosts that vectorize the infection among mammals. This parasite oxidizes proline to glutamate through two enzymatic steps and one nonenzymatic step. In insect vectors, T. cruzi differentiates from a noninfective replicating form to nonproliferative infective forms. Proline sustains this differentiation, but to date, a link between proline metabolism and differentiation has not been established. In T. cruzi, the enzymatic steps of the proline-glutamate oxidation pathway are catalysed exclusively by the mitochondrial enzymes proline dehydrogenase [TcPRODH, EC: 1.5.5.2] and D1-pyrroline-5-carboxylate dehydrogenase [TcP5CDH, EC: 1.2.1.88]. Both enzymatic steps produce reducing equivalents that are able to directly feed the mitochondrial electron transport chain (ETC) and thus produce ATP. In this study, we demonstrate the contribution of each enzyme of the proline-glutamate pathway to ATP production. In addition, we show that parasites overexpressing these enzymes produce increased levels of H2O2, but only those overexpressing TcP5CDH produce increased levels of superoxide anion. We show that parasites overexpressing TcPRODH, but not parasites overexpressing TcP5CDH, exhibit a higher rate of differentiation into metacyclic trypomastigotes in vitro. Finally, insect hosts infected with parasites overexpressing TcPRODH showed a diminished parasitic load but a higher percent of metacyclic trypomastigotes, when compared with controls. Our data show that parasites overexpressing both, PRODH and P5CDH had increased mitochondrial functions that orchestrated different oxygen signalling, resulting in different outcomes in relation to the efficiency of parasitic differentiation in the invertebrate host.

scholarly journals A Comprehensive Assessment of Apigenin as an Antiproliferative, Proapoptotic, Antiangiogenic and Immunomodulatory Phytocompound

Nutrients ◽  
2019 ◽  
Vol 11 (4) ◽  
pp. 858 ◽  
Author(s):  
Alexandra Ghițu ◽  
Anja Schwiebs ◽  
Heinfried H. Radeke ◽  
Stefana Avram ◽  
Istvan Zupko ◽  
...  
Keyword(s):  
Comprehensive Evaluation ◽  
Annexin V ◽  
Dependent Manner ◽  
Experimental Conditions ◽  
Cell Growth And Migration ◽  
Atp Production ◽  
Lactate Dehydrogenase Release ◽  
High Concentrations ◽  
And Migration ◽  
Lps Activation

Apigenin (4′,5,7-trihydroxyflavone) (Api) is an important component of the human diet, being distributed in a wide number of fruits, vegetables and herbs with the most important sources being represented by chamomile, celery, celeriac and parsley. This study was designed for a comprehensive evaluation of Api as an antiproliferative, proapoptotic, antiangiogenic and immunomodulatory phytocompound. In the set experimental conditions, Api presents antiproliferative activity against the A375 human melanoma cell line, a G2/M arrest of the cell cycle and cytotoxic events as revealed by the lactate dehydrogenase release. Caspase 3 activity was inversely proportional to the Api tested doses, namely 30 μM and 60 μM. Phenomena of early apoptosis, late apoptosis and necrosis following incubation with Api were detected by Annexin V-PI double staining. The flavone interfered with the mitochondrial respiration by modulating both glycolytic and mitochondrial pathways for ATP production. The metabolic activity of human dendritic cells (DCs) under LPS-activation was clearly attenuated by stimulation with high concentrations of Api. Il-6 and IL-10 secretion was almost completely blocked while TNF alpha secretion was reduced by about 60%. Api elicited antiangiogenic properties in a dose-dependent manner. Both concentrations of Api influenced tumour cell growth and migration, inducing a limited tumour area inside the application ring, associated with a low number of capillaries.

Sign in / Sign up

Export Citation Format

Share Document