Unique PFK regulatory property from some mosquito vectors of disease, and from Drosophila melanogaster

Abstract

BackgroundArthropod-borne diseases are some of the most rapidly spreading diseases. Reducing the vector population is currently the only effective way to reduce case numbers. Central metabolic pathways are potential targets to control vector populations, but have not been well explored to this aim. The information available on energy metabolism, as a way to control lifespan and dispersion through flight of dipteran vectors, is inadequate.MethodsPhosphofructokinase (PFK) activity was measured in the presence of both of its substrates, fructose-6-phosphate (F6P) and ATP, as well as some allosteric effectors: Fructose- 2,6 – bisphosphate (F2, 6BP), citrate and AMP. Aedes aegypti phosphofructokinase sequence (AaPFK) was aligned with many other insects and also vertebrate sequences. A 3D AaPFK model was produced and docking experiments were performed with AMP and citrate.ResultsThe kinetic parameters of AaPFK were determined for both substrates: F6P (V = 4.47 ± 0.15 μmol of F1, 6BP/min, K0.5 = 1.48 ± 0.22 mM) and ATP (V = 4.73 ± 0.57 μmol of F1, 6BP/min, K0.5 = 0.43 ± 0.10 mM). F2,6P was a powerful activator of AaPFK, even at low ATP concentrations. AaPFK inhibition by ATP was not enhanced by citrate, consistent with observations in other insects. After examining the sequence alignment of insect and non-insect PFKs, the hypothesis is that a modification of the citrate binding site is responsible for this unique behavior. AMP, a well-known positive effector of PFK, was not capable of reverting ATP inhibition. Aedes, Anopheles and Culex are dengue, malaria and filariasis vectors, respectively, and are shown to have this distinct characteristic in phosphofructokinase control. The alignment of several insect PFKs suggested a difference in the AMP binding site and a significant change in local charges, which introduces a highly negative charge in this part of the protein, making the binding of AMP unlikely. This hypothesis was supported by 3D modeling of PFK with AMP docking, which suggested that the AMP molecule binds in a reverse orientation due to the electrostatic environment. The present findings imply a potential new way to control PFK activity and are a unique feature of these Diptera.ConclusionsThe present findings provide the first molecular explanation for citrate insensitivity in insect PFKs, as well as demonstrating for the first time AMP insensitivity in dipterans. It also identified a potential target for novel insecticides for the control of arthropod-borne diseases.Electronic supplementary materialThe online version of this article (doi:10.1186/s13071-016-1391-y) contains supplementary material, which is available to authorized users.

A Drosophila model of combined D-2- and L-2-hydroxyglutaric aciduria reveals a mechanism linking mitochondrial citrate export with oncometabolite accumulation

“…Such a model would also explain why citrate treatment could reduce 2HG levels in a patient with combined D-/L-2HGA ( Muhlhausen et al, 2014 ), as partial restoration of cytosolic citrate levels would inhibit PFK enzyme activity and reduce glycolytic flux ( Kemp and Gunasekera, 2002 ). Although it is tempting to speculate that citrate also inhibits Drosophila PFK, we would note that insect PFK homologs, when isolated from adult muscle tissue, are not inhibited by citrate ( Nunes et al, 2016 ). Regardless of the mechanism, our findings demonstrate that citrate governs glycolytic flux in Drosophila larvae.…”

Polyphenol-Rich Diets Exacerbate AMPK-Mediated Autophagy, Decreasing Proliferation of Mosquito Midgut Microbiota, and Extending Vector Lifespan

“…Vectorial capacity, the ability of mosquitoes to transmit parasites or pathogens, depends on several factors, including vector competence, mosquito population density, flight capacity, host preferences, biting rate, mosquito immunity and lifespan [ 4 – 6 ]. Aedes aegypti is the most important global vector of dengue viruses.…”

Anopheles metabolic proteins in malaria transmission, prevention and control: a review

“…absent in non-target species). Different studies have suggested some metabolic proteins that can serve as possible insecticide targets, namely, carbonic anhydrases, arylalkylamine N-acetyltransferases, V-ATPase and phosphofructokinase (PFK) [49][50][51][52]. These potential insecticide targets are further discussed in the subsection "Identifying novel insecticide targets".…”

“…Phosphofructokinase (PFK, EC 2.7.1.11), a key regulatory enzyme, catalyzing the committed step in the glycolytic pathway has been proposed as a potential insecticide target [50]. In the experiments carried out by Nunes et al [50], it was observed that PFK inhibition by ATP in Ae. aegypti was not enhanced by citrate, and AMP could not relieve ATP inhibition of PFK.…”

“…In addition, amino acid substitution in AMP binding sites resulted in changes in the overall electrostatic charges of insect PFKs compared to that of humans. These substitutions in AMP and citrate binding sites make PFK in these insects insensitive to regulation by citrate and AMP [50]. Considering the importance of PFK for energy metabolism and consequent survival, these insect unique modifications in PFK could be exploited in disease vectors to produce highly specific and selective insecticides.…”