Jacky Snoep

Up-regulation of glycolytic flux (glucose to lactate conversion), also known as the “Warburg effect”, has been associated with 90% of cancers. We implemented a systems biology approach in order to improve the overall understanding of the “Warburg effect” as well as identifying drug targets within the glycolytic pathway.

Hypoglycaemia and lactic acidosis are key diagnostics for poor chances of survival in malaria patients. In this project we aim to test to what extent the metabolic activity of Plasmodium falciparum contributes to a changed glucose metabolism in malaria patients. The approach is to start with detailed bottom up models for the parasite and then merge these with more coarse grained models at the whole body level.

The Molecular Systems Biology project holds information for reproducing simulation figures in the journal. This can include experimental data files, model files and manuscript information.

The goal of the project is to establish a new biotechnological platform for the production of hydroxy-amino acids, since the current production of these important building blocks is very expensive. Enzyme engineering, systems biotechnology and metabolic engineering will be used in a synthetic biology approach.

LEADER: IPF (prof. A. Lederer)

PARTNERS: IPF & StU

OBJECTIVES:

• Determination of stability, degradation and drug release kinetics.

• Mathematical modelling of response to treatments of infectious diseases.

DELIVERABLES

10.2022 M 3.1 ...

Background- Thermophilic organisms are composed of both bacterial and archaeal species. The enzymes isolated from these species and from other extreme habitats are more robust to temperature, organic solvents and proteolysis. They often have unique substrate specificities and originate from novel metabolic pathways. Thermophiles as well as their stable enzymes (‘thermozymes’) are receiving increased attention for biotechnological applications.

The proposed project will establish thermophilic in ...

Cyanobacteria are the only prokaryotes performing oxygenic photosynthesis. Theyhad and have tremendous influence on the biogeochemical cycles on Earth. Recently,cyanobacteria are increasingly investigated as cell factories for a sustainableeconomy. Despite their global, environmental and rising economic importance, ourknowledge on the regulation of their primary metabolism is fragmented.Cyanobacteria switch between photoautotrophic and heterotrophic modes ofmetabolism during day/night cycles or ...

FAIRDOM will establish a support and service network for European Systems Biology. We will serve projects in standardising, managing and disseminating data and models in a FAIR manner: Findable, Accessible, Interoperable and Reusable.

Silicon cell model for the central carbohydrate metabolism of the archaeon Sulfolobus solfataricus under temperature variation

The main objectives of SysMO-DB are to: facilitate the web-based exchange of data between research groups within- and inter- consortia, and to provide an integrated platform for the dissemination of the results of the SysMO projects to the scientific community. We aim to devise a progressive and scalable solution to the data management needs of the SysMO initiative, that:

• facilitates and maximises the potential for data exchange between SysMO research groups;
• maximises the ‘shelf life’ and ...

Within the e:Bio - Innovationswettbewerb Systembiologie (Federal Ministry of Education and Research (BMBF)), the SulfoSYSBIOTECH consortium (10 partners), aim to unravel the complexity and regulation of the carbon metabolic network of the thermoacidophilic archaeon Sulfolobus solfataricus (optimal growth at 80°C and pH 3) in order to provide new catalysts ‘extremozymes’ for utilization in White Biotechnology.

Based on the available S. solfataricus genome scale metabolic model (Ulas et al., 2012) ...

Comparative Systems Biology: Lactic Acid Bacteria

Systems analysis of process-induced stresses: towards a quantum increase in process performance of Pseudomonas putida as the cell factory of choice for white biotechnology.

The specific goal of this project is to exploit the full biotechnological efficacy of Pseudomonas putida KT2440 by developing new optimization strategies that increase its performance through a systems biology understanding of key metabolic and regulatory parameters that control callular responses to key stresses generated ...

MOSES (Micro Organism Systems biology: Energy and Saccharomyces cerevisiae) develops a new Systems Biology approach, which is called 'domino systems biology'. It uses this to unravel the role of cellular free energy ('ATP') in the control and regulation of cell function. MOSES operates though continuous iterations between partner groups through a new systems-biology driven data-management workflow. MOSES also tries to serve as a substrate for three or more other SYSMO programs.

The oxidative Weimberg pathway for the five-step pentose degradation to α ketoglutarate from Caulobacter crescentus is a key route for sustainable bioconversion of lignocellulosic biomass to added-value products and biofuels. Here, we developed a novel iterative approach involving initial rate kinetics, progress curves, and enzyme cascades, with high resolution NMR analysis of intermediate dynamics, and multiple cycles of kinetic modelling analyses to construct and validate a quantitative model ...

Biphasic response as a mechanism against mutant takeover in tissue homeostasis circuits

Frequency doubling in the cyanobacterial circadian clock

The gluconeogenic conversion of 3-phosphoglycerate via 1,3-bisphosphoglycerate to glyceraldehyde-3-phosphate was compared at 30 C and at 70 C. At 30 C it was possible to produce 1,3-bisphosphoglycerate from 3-phosphoglycerate with phosphoglycerate kinase, but at 70 C, 1,3- bisphosphoglycerate was dephosphorylated rapidly to 3-phosphoglycerate, effectively turning the phosphoglycerate kinase into a futile cycle. At both temperatures it was possible to convert 3-phosphoglycerate to glyceraldehyde ...

Drug detoxification dynamics explain the postantibiotic effect

Protein abundance of AKT and ERK pathway components governs cell-type- specific regulation of proliferation

The investigation entails the construction and validation of a detailed mathematical model for glycolysis of the malaria parasite Plasmodium falciparum in the blood stage trophozoite form.

Design principles of nuclear receptor signaling: how complex networking improves signal transduction

Division of labor by dual feedback regulators controls JAK2/STAT5 signaling over broad ligand range

An investigation in the central carbon metabolism of S. solfataricus with a focus on the unique temperature adaptations and regulation; using a combined modelling and experimental approach.

One pot cascade - pathway analysis for the purified Caulinobacter crescentus Weimberg pathway enzymes. Effect of co-factor recycling, removal of XLA, and optimisation on Xylose to aKG is studied.

https://jjj.bio.vu.nl/models/experiments/shen2020_fig3a/simulate https://jjj.bio.vu.nl/models/experiments/shen2020_fig3b/simulate https://jjj.bio.vu.nl/models/experiments/shen2020_fig3c/simulate https://jjj.bio.vu.nl/models/experiments/shen2020_fig3d/simulate

Initial rate kinetics for the purified Caulinobacter crescentus Weimberg pathway enzymes, including substrate dependence, and product inhibition.

Progress curves for the purified Caulinobacter crescentus Weimberg pathway enzymes. Each reaction is followed up to completion and then the next enzyme in the pathway is added, i.e. XDH-XLA-XAD-KDXD and finally KGSADH

Cell free extract - pathway analysis for Caulinobacter crescentus Weimberg pathway enzymes. Effect of co-factor recycling, and Mn2+ on Xylose to aKG conversion is studied.

D Biphasic control of stem-cell expansion, where stem-cell expansion is low both at high and low concentrations of y. The system has a stable fixed point at the concentration of y where pr = 0.5 and an unstable fixed point at some lower concentration of y.

SED-ML simulation https://jjj.bio.vu.nl/models/experiments/karin2017_fig4d/simulate

C A mutated stem cell with a strong inactivation of the sensing of y has a growth advantage (differentiates less), and therefore, it invades the stem- cell population. As a result, both the stem-cell pool and the number of terminally differentiated cells increase.

SED-ML simulation https://jjj.bio.vu.nl/models/experiments/karin2017_fig4c/simulate

Mathematical simulation of a tamoxifen-induced conditional knock-in of a sixfold activating GCK mutant in beta cells. (C) The percentage of beta cells with mutated GCK increases to ~25% after 3 days, but then decreases and is eliminated after a few weeks. (D) Glucose levels initially decrease after the tamoxifen injection, but return to normal after a few weeks. Insets: Experimental results of Tornovsky-Babeay et al (2014).

SED-ML simulation https://jjj.bio.vu.nl/models/experiments/karin2017_fig2/simulate ...

C Trajectories of Z from different initial concentrations of cells (Z) (i) or y (ii) for the circuit of (B). The healthy concentration Z = ZST is reached regardless of initial concentration of Z, as long as it is nonzero, and regardless of the initial concentration of y.

SED-ML simulation https://jjj.bio.vu.nl/models/experiments/karin2017_fig1c/simulate

D An arrow marks the time when a mutant with a strong activation of the sensing of y arises (for the circuit depicted in B). This mutant has a selective advantage and takes over the population.

SED-ML simulation https://jjj.bio.vu.nl/models/experiments/karin2017_fig1d/simulate

G Trajectories of Z from different initial concentrations of Z (i) or y (ii) for the circuit depicted in (F). The healthy concentration Z = ZST is not reached for small values of Z (Z << ZST) or large values of y (y >> yUST).

SED-ML simulation https://jjj.bio.vu.nl/models/experiments/karin2017_fig1g/simulate

H The arrows mark the times when a mutant with a strong activation of the sensing of y arises (for the biphasic circuit depicted in F). This mutant has a selective disadvantage and is thus eliminated.

SED-ML simulation https://jjj.bio.vu.nl/models/experiments/karin2017_fig1h/simulate

C Numerical simulations of the RpoD6 wild-type network show a shoulder of expression trailing the main peak (red line). All the parameters describing the clock and SigC are as in Fig 4B, and only the threshold of activation of the rpoD6 promoter by the clock was modified. Numerical simulations of a SigC knock-out model (in which the terms representing the regulation of RpoD6 by SigC are set to zero) show only single-peaked oscillations (blue line). D The incoherent feedforward loop circuit that ...

Numerical simulations of the wild-type network show double peaks of expression (red line), and numerical simulations of a SigC knock-out model (in which the terms representing the regulation of PsbAI by SigC are set to zero) show only single-peaked oscillations (blue line)..

SED-ML simulation https://jjj.bio.vu.nl/models/experiments/martins2016_fig4b/simulate

Kinetic characterisation of phosphoglycerate kinase

Mathematical model for PGK kinetics, saturation with ADP, ATP, 3PG and BPG.

Kinetic characterisation of glyceraldehyde 3-phosphate dehydrogenase

In vitro reconstitution of the PGK, GAPHD, TPI and FBPAase enzymes from S. solfataricus

Model prediction of the conversion of 3PG to fructose-6-phosphate and the gluconeogenic pathway intermediates. https://jjj.bio.vu.nl/models/experiments/kouril3_experiment-user/simulate

Mathematical model for GAPDH kinetics, saturation with BPG, NADPH, NADP, GAP and Pi

Kinetic characterisation of triose phosphate isomerase

Mathematical model for TPI kinetics, saturation with GAP and DHAP, and inhibition by 3PG and PEP

Kinetic characterisation of fructose 1,6-bisphosphate aldolase phosphatase

Mathematical model for FBPAase kinetics, saturation with DHAP and GAP

Temperature degradation of BPG, GAP and DHAP

Modelling the degradation of BPG, GAP and DHAP at high temperature

Experimental data for the conversion of 3PG to F6P and the gluconeogenic pathway intermediates

SED-ML simulation: https://jjj.bio.vu.nl/models/experiments/bachmann2011/simulate

SED-ML simulation: https://jjj.bio.vu.nl/models/experiments/bachmann2011/simulate

Experimental data for the yeast PGK incubations at 30C, with and without recycling of ATP.

Data files for the conversion of XYL to KG, via the sequential addition of the Caulobacter crescentus Weimberg pathway enzymes, XDH, XLA, XAD, KDXD, KGSADH.

Initial rate kinetics for the α-ketoglutarate semialdehyde dehydrogenase of Caulobacter crescentus, α-ketoglutarate semialdehyde and NAD saturation, and α-ketoglutarate, NADH and 2-keto-3-deoxy-D-xylonate inhibition.

Initial rate kinetics for the 2-keto-3-deoxy-D-xylonate dehydrates of Caulobacter crescentus, 2-keto-3-deoxy-D-xylonate saturation and inhibitor titrations.

Initial rate kinetics for the xylonate dehydratase of Caulobacter crescentus, xylonate saturation.

Initial rate kinetics for the xylonolactonase reaction of Caulobacter crescentus, xylonolactone saturation for the enzyme catalysed reaction, and for the non-enzymatic reaction.

Initial rate kinetics for xylose dehydrogenase of Caulobacter crescentus, saturation with xylose and NAD, and inhibition by NADH and xylonolactone.

Data files for the conversion of XYL to KG, via the Caulobacter crescentus Weimberg pathway, using old enzymes: XDH, XLA, XAD, KDXD, KGSADH, with NAD recycling, and optimal protein distribution.

Data files for the conversion of XYL to KG, in Caulobacter crescentus cell free extract, with NAD recycling, and additional Mn2+ (0.15 mM) added.

Data files for the conversion of XYL to KG, in Caulobacter crescentus cell free extract, without NAD recycling, but with additional Mn2+ (0.15 mM) added.

Data files for the conversion of XYL to KG, in Caulobacter crescentus cell free extract, with NAD recycling, but without additional Mn2+ added.

Data files for the conversion of XYL to KG, via the Caulobacter crescentus Weimberg pathway enzymes, XDH, XLA, XAD, KDXD, KGSADH.

Data files for the conversion of XYL to KG, via the Caulobacter crescentus Weimberg pathway enzymes, omitting XLA: XDH, XAD, KDXD, KGSADH.

Data files for the conversion of XYL to KG, via the Caulobacter crescentus Weimberg pathway enzymes: XDH, XLA, XAD, KDXD, KGSADH, with NAD recycling.

Experimental data for 3PG conversion to fructose-6-phosphase in reconstituted systems of gluconeogenesis of S. solfataricus

Simulation results of TPI experimental data for GAP and DHAP saturation.

Simulation results of temperature degradation of gluconeogenic intermediates

Simulation results of experimental data of the reconstituted gluconeogenic system

Simulation results of PGK experimental data for ADP, ATP, 3PG and BPG saturation.

Simulation results of FBPAase of experimental data for DHAP and GAP saturation

Model for the Caulobacter crescentus Weimberg pathway, describing the conversion of Xyl to KG upon sequential adition of purified enzymes. If the Mathematica notebook is downloaded and the data file is downloaded in the same directory, then the notebook can be evaluated, and the figure in the manuscript for the progress curves will be reproduced.

Model for the Caulobacter crescentus Weimberg pathway, describing the conversion of Xyl to KG upon sequential adition of purified enzymes. If the Mathematica notebook is downloaded and the data file is downloaded in the same directory, then the notebook can be evaluated, and the figure in the manuscript for the progress curves will be reproduced.

Model for the Caulobacter crescentus Weimberg pathway, describing the conversion of Xyl to KG upon sequential adition of purified enzymes. If the Mathematica notebook is downloaded and the data file is downloaded in the same directory, then the notebook can be evaluated, and the figure in the manuscript for the progress curves will be reproduced.

Model for the Caulobacter crescentus Weimberg pathway, describing the conversion of Xyl to KG upon sequential adition of purified enzymes. If the Mathematica notebook is downloaded and the data file is downloaded in the same directory, then the notebook can be evaluated, and the figure in the manuscript for the progress curves will be reproduced.

Model for the Caulobacter crescentus Weimberg pathway, describing the conversion of Xyl to KG upon sequential adition of purified enzymes. If the Mathematica notebook is downloaded and the data file is downloaded in the same directory, then the notebook can be evaluated, and the figure in the manuscript for the progress curves will be reproduced.

Model for the Caulobacter crescentus Weimberg pathway, describing the conversion of Xyl to KG upon sequential adition of purified enzymes. If the Mathematica notebook is downloaded and the data file is downloaded in the same directory, then the notebook can be evaluated, and the figure in the manuscript for the progress curves will be reproduced.

Model for the Caulobacter crescentus Weimberg pathway, describing the conversion of Xyl to KG in cell free extract. If the Mathematica notebook is downloaded and the data file is downloaded in the same directory, then the notebook can be evaluated, and the figure in the manuscript for the cell free extract with added Mn, but no NAD rec, will be reproduced.

Model for the Caulobacter crescentus Weimberg pathway, describing the conversion of Xyl to KG. If the Mathematica notebook is downloaded and the data file is downloaded in the same directory, then the notebook can be evaluated, and the figure in the manuscript for cascade 12 will be reproduced.

Model for the Caulobacter crescentus Weimberg pathway, describing the conversion of Xyl to KG in cell free extract. If the Mathematica notebook is downloaded and the data file is downloaded in the same directory, then the notebook can be evaluated, and the figure in the manuscript for the cell free extract with no added Mn, but with NAD rec, will be reproduced.

Steady state model for the Caulobacter crescentus Weimberg pathway, describing the conversion of Xyl to KG. Protein levels need to be adapted to CFE levels, see SED-ML scripts.

Model for the Caulobacter crescentus Weimberg pathway, describing the conversion of Xyl to KG, with NAD recycling. If the Mathematica notebook is downloaded and the data file is downloaded in the same directory, then the notebook can be evaluated, and the figure in the manuscript for cascade 13 will be reproduced.

Model for the Caulobacter crescentus Weimberg pathway, describing the conversion of Xyl to KG. If the Mathematica notebook is downloaded and the data file is downloaded in the same directory, then the notebook can be evaluated, and the figure in the manuscript for cascade 10 will be reproduced.

Model for the Caulobacter crescentus Weimberg pathway, describing the conversion of Xyl to KG, using old enzymes, with optimal protein distribution. If the Mathematica notebook is downloaded and the data file is downloaded in the same directory, then the notebook can be evaluated, and the figure in the manuscript for cascade 16 will be reproduced.

Model for the Caulobacter crescentus Weimberg pathway, describing the conversion of Xyl to KG.

Model for the Caulobacter crescentus Weimberg pathway, describing the conversion of Xyl to KG in cell free extract. If the Mathematica notebook is downloaded and the data file is downloaded in the same directory, then the notebook can be evaluated, and the figure in the manuscript for the cell free extract with added Mn and NAD rec will be reproduced.

Model for the Caulobacter crescentus Weimberg pathway, describing the conversion of Xyl to KG. Protein levels need to be adapted to CFE levels, see SED-ML scripts

Model for the Caulobacter crescentus α-ketoglutarate semialdehyde dehydrogenase, describing the initial rate kinetics for substrate dependence and product inhibition. If the Mathematica notebook is downloaded and the data file for the XAD kinetics is downloaded in the same directory, then the notebook can be evaluated. The model in the notebook will then be parameterised and the figures in the manuscript for KGSADH will be reproduced.

Model for the Caulobacter crescentus Weimberg pathway, describing the conversion of Xyl to KG, with sequential addition of purified enzymes.

Model for the Caulobacter crescentus xylose dehydrogenase, describing the initial rate kinetics including substrate dependence and product inhibition. If the Mathematica notebook is downloaded and the data file for the XDH kinetics is downloaded in the same directory, then the notebook can be evaluated. The model in the notebook will then be parameterised and the figures in the manuscript for XDH will be reproduced.

Model for the Caulobacter crescentus xylonolactonase, describing the initial rate kinetics and substrate dependence. If the Mathematica notebook is downloaded and the data file for the XLA kinetics is downloaded in the same directory, then the notebook can be evaluated. The model in the notebook will then be parameterised and the figures in the manuscript for XLA will be reproduced.

SOP for the determination of external metabolites (Glc, Pyr, Gly, Lac) in intact trophozoite incubations, and for the determination of intracellular metabolite concentrations.

SOP for the cultivation conditions of Plasmodium falciparum, including the protocol for synchronisation.

SOP for measurement of G3PDH activity in extracts.

SOP for measurement of ALD activity in extracts.

SOP for measurement of glucose transport activity in intact trophozoites.

SOP for measurement of GAPDH activity in extracts.

SOP for measurement of ENO activity in extracts.

SOP for measurement of HK activity in extracts.

SOP for measurement of PFK activity in extracts.

SOP for measurement of LDH activity in extracts.

SOP for measurement of PGK activity in extracts.

SOP for measurement of PK activity in extracts.

SOP for measurement of PGM activity in extracts.

SOP for measurement of PGI activity in extracts.

SOP for measurement of PGM activity in extracts.

SOP for measurement of TPI activity in extracts.

SOP for the isolation of intact Plasmodium falciparum trophozoites from infected red blood cells and the preparation of a cell free extract that can be used for kinetic analyses.

Preparation of cell free extracts of the recombinant E. coli strains expressing the gluconeogenic S. solfataricus enzymes.

Cloning and heterologous expression of gluconeogenic enzymes from S. solfataricus in E. coli

Purification of gluconeogenic enzymes from S. solfataricus in recombinant E.coli extracts