The role of a positive feedforward loop in regulating yeast glycolytic intermediate dynamics

Kotze, Sone (2021-12)

Thesis (MSc)--Stellenbosch University, 2021.

Thesis

ENGLISH ABSTRACT: Yeast glycolysis is a well-studied model for metabolic pathways and fundamental aspects of regulation. Whereas negative feedback is common in metabolic networks; positive feedforward regulation is much less often observed. The aim of this project was to elucidate the function of feedforward loops (FFLs); investigating why they occur and to discern their potential role in disease states. A well-known FFL in glycolysis was investigated, namely that of pyruvate kinase (PK) which is known to be strongly regulated. One of its most important activators is fructose 1,6-bisphosphate (FBP), a metabolite found upstream in the pathway. Certain cancer cell lines have been shown to induce a particular isoform of PK, PKM2, which is not activated by FBP, hinting at an advantage that the absence of this FFL may confer to cancerous cells. By defining the fundamental function of FFLs, greater insights into metabolism can be obtained by investigating the effects of its absence on glycolytic flux. To probe this question, we used a systems biology approach of experimentation in conjunction with mathematical modelling. Yeast PK was kinetically characterised using cell-free extracts and was found to be activated by FBP via an FFL and inhibited by inorganic phosphate (Pi). This kinetic data was used to analyse several PK rate equations that incorporate allosteric models for its regulation by FBP and Pi. Based on these results a Hill-type equation was selected to extend an existing detailed kinetic model for yeast glycolysis. In addition, core model simulations were performed on systems with increasing complexity to formulate the core model hypothesis that the function of FFLs in metabolic networks is to buffer the intermediate metabolites between the regulating metabolite and regulated reaction when flux through the pathway changes, thereby preventing metabolic imbalance. We tested this hypothesis via experimental analysis of yeast glycolytic intermediates and cofactors over time after a glucose pulse to the pathway. The results showed interesting cofactor dynamics; with ATP being fully converted to AMP during glucose exhaustion and also indicated that Pi could not function as a switch-off mechanism for the FFL. These glycolytic intermediate dynamics also served as an independent experimental data-set that successfully validated the full glycolytic model that was adapted with the Hill equation to better describe PK activity and flux through the pathway as a whole.

AFRIKAANSE OPSOMMING: Gisglikolise is ’n goed bestudeerde model vir metaboliese paaie en die fundamentele aspekte van regulering. Negatiewe terugvoer is algemeen in metaboliese netwerke en is dus goed bestudeer, maar positiewe terugvoerregulering word selde waargeneem. Die doel van hierdie projek is om die funksie van positiewe voorwaartse lusse (FFLs) uit te pluis; om te ondersoek waarom dit voorkom, èn om hul potensiële rol in siektetoestande te onderskei. ’n Bekende voorwaartse lus in glikolise word ondersoek, díe van pyruvatkinase (PK), ’n ensiem wat sterk gereguleer word. Een van die belangrikste aktiveerders van PK is fruktose 1,6-bisphopshate (FBP), ’n metaboliet wat stroomop in die pad voorkom. Daar is bewys dat sekere kankersellyne ’n spesifieke isoform van PK, PKM2, uitdruk, wat nie deur FBP geaktiveer word nie. Dit dui moontlik op ’n voordeel wat die afwesigheid van die voorwaartse lus aan kankerselle kan bestee. Deur die fundamentele funksie van voorwaartse lusse te definieer kan ons beter insigte kry oor metabolisme, deur die uitwerking van die afwesigheid daarvan op glikolitiese vloed te ondersoek. Om hierdie vraag te ondersoek, het ons ’n stelselbiologie-benadering van eksperimentering in kombinasie met wiskundige modellering gebruik. Gis-PK is kineties gekarakteriseer deur gebruik te maak van selvrye gisekstrakte en daar is bevind dat dit deur FBP via ’n FFL geaktiveer is, en deur anorgaiv niese fosfaat (Pi) geïnhibeer word. Hierdie kinetiese data is toe gebruik om PK-koersvergelykings te analiseer wat allosteriese modelle inkorporeer vir die regulering van PK deur FBP en Pi. Op grond van hierdie resultate is ’n Hill-tipe vergelyking gekies om ’n bestaande gedetailleerde kinetiese model vir gisglikolise uit te brei. Verder het ons speelgoedmodelsimulasies uitgevoer op stelsels met toenemende kompleksiteit om die kernmodelhipotese te formuleer dat die funksie van FFLs in metaboliese netwerke is om die intermediêre metaboliete tussen die regulerende en gereguleerde metaboliet te buffer wanneer vloei deur die pad verander en sodoende help om metaboliese wanbalans te ontmoedig. Ons het hierdie hipotese getoets deur middel van die ontleding van gisglikolitiese tussenprodukte en ko-faktore oor tyd na ’n glukosepuls. Die eksperimente het interessante kofaktordinamika getoon met ATP wat byna ten volle omgeskakel is na AMP tydens glukose-uitputting. Met hierdie resultate het ons ook Pi uitgesluit as ’n moontlike afskakel meganisme vir die PK-FFL. Hierdie glikolitiese intermediêre dinamika het ook gedien as ’n onafhanklike eksperimentele datastel wat die volledige glikolitiese model- wat met die Hillvergelyking aangepas is- suksesvol gevalideer het in ’n poging om PK-aktiwiteit en vloei deur die hele pad beter te beskryf.

Please refer to this item in SUNScholar by using the following persistent URL: http://hdl.handle.net/10019.1/124260
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