Phosphofructokinase isoforms as metabolic targets for treating neurological diseases
Fernandes, Peter Mark
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The breakdown of glucose to pyruvate, known as glycolysis, is a central biochemical pathway, critically important for energy production and biosynthesis. Phosphofructokinase (PFK), the third enzyme in the pathway, is a crucial regulator of glycolytic flux, being the first committed step of glycolysis and modulating entry into the pentose-phosphate-pathway. Alterations in PFK activity have been implicated in many neurological conditions, including Tarui’s disease, epilepsy, Alzheimer’s disease, Down’s syndrome, and cancers. There are three isoforms of human PFK; it is assumed that these evolved to fulfil specific metabolic niches both within cells and between tissue types. However, the differences between isoforms have never been systematically compared. Understanding these differences is an essential prerequisite for developing novel therapeutic agents targeting human PFK. Trypanosomatid parasites are a major global cause of neurological morbidity and mortality. Neglected tropical diseases caused by trypanosomatid parasites include African Sleeping Sickness (Trypanosoma brucei), Chagas disease (Trypanosoma cruzi), and leishmaniasis (Leishmania spp.). There is increasing interest in targeting the metabolic enzymes of these parasites, including PFK, which greatly differ from mammalian counterparts. This thesis describes biochemical, bio-physical, and bio-informatic studies on the three human PFK isoforms (PFK-M, PFK-L, and PFK-P), expressed in S. cerevisiae. Biophysical studies showed that the active conformation was tetrameric, with activity regulated by time and concentration dependent dissociation into smaller inactive species. The propensity to dissociate differed between isoforms, with PFK-M being most stable and PFK-P least stable. Dissociation was synergistically slowed by the addition of substrates and reducing agents, indicating different mechanisms of action. Kinetic studies were performed with respect to both substrates (ATP and F6P) in the presence of natural metabolites hypothesised to act as modulators of enzyme activity. Each isoform conformed to an allosteric sigmoidal kinetic model and had differing kinetic properties, with PFK-M being the most active and PFK-P the least active. ATP was found to act as both substrate and allosteric inhibitor, with activity showing a biphasic response to ATP concentration. Each isoform showed different susceptibilities to both ATP inhibition and regulation by allosteric modulators. The reverse reaction was shown to be possible under certain conditions. Bio-informatic data on intra-cellular and inter-cellular locations were determined using the Human Protein Atlas and the FANTOM5 datasets. PFK-M localises to the cytosol and may co-localise with endoplasmic reticulum; PFK-L associates with nucleoli and mitochondria; and PFK-P is cytosolic. Splice variants were not shown to be physiologically significant. Each isoform had different tissue expression levels, with overall PFK expression varying by tissue type. PFK-P was the principal isoform in cancers, whereas PFK-L was dominantly expressed in immune cells. Activated macrophages switched rapidly from PFK-L to PFK-P. PFK-M and PFK-P were the dominant isoforms in the brain, although there were differences between brain areas. Neurons expressed less PFK than astrocytes, in keeping with the lactate shuttle theory. PFK from each of the three main pathological trypanosomatid species were compared (T. brucei, TbPFK; T. cruzi, TcPFK; L. infantum, LmPFK); expressed in E. coli. Biophysical analysis showed each PFK to be tetrameric; no evidence of time or concentration dependent dissociation or inactivation was found. Kinetic properties differed between isoforms, with TcPFK being most active and LmPFK being least active. LmPFK was very poorly active with regard to F6P titrations unless AMP was present. No other modulators were shown to affect activity, although GTP was an alternate substrate. The reverse reaction was shown to be possible and may be compatible with physiological concentrations of ADP and F16BP in the trypanosomatid glycosome.