Figure 1.
Schematic overview of the models used for simulation.
(A) Detailed scheme of the modeled metabolic pathways. The numbered arrows correspond to reactions from Table 1. Extensions to the original model of glycolysis are indicated by colored shapes. Boundary metabolites are in bold, glycosomal Rib-5-P is a boundary metabolite in model C and D. (B) Schematic overview of the different models, each consisting of a unique combination of the colored modules described in (A) and Table 1. Model C and D can alternatively utilize fructose (model Cfru and Dfru), but this branch is switched off unless specifically mentioned.
Table 1.
Model stoichiometry by modules and their subsequent reactions.
Figure 2.
Phosphate leak in model with PPP.
Time course simulation of model B, in which the reactions of the glycosomal PPP are switched on at t = 0 by increasing their Vmax value from zero to the value given in Table S1. Glce is 5 mM and kTOX = 2 µl·min−1·mg protein−1. Solid lines indicate medians, shaded areas show interquartile ranges. Fluxes (J) are plotted on the left y-axis and are indicative of glucose uptake (GlcTplasma membrane), glycerol (GK) and pyruvate production (PyrT) and the two branches of pentose phosphate pathways (G6PDHc/g). The sum of bound phosphates in the glycosome (ΣPg), as exists in the model of glycolysis (Table 2, moiety 5), is plotted on the right y-axis. Within 25 minutes, all bound phosphates within the glycosome are depleted and all metabolic fluxes subsequently drop to zero.
Table 2.
Conserved moieties in the four models of Figure 1.
Figure 3.
ATP:ADP antiporter mimics turbo-state.
(A) Overview of the models used in this figure. Model A and D are from Figure 1, model A–glyc is model A without glycosomal localization, as described in [31], model A+AAT is model A with an ATP:ADP antiporter. (B–C) Steady-state concentrations of glycosomal Glc-6-P and Fru-1,6-BP are depicted in the various models. (D) Increasing the activity of the ATP:ADP antiporter (Vmax,ATP:ADP antiporter) in model D leads to a high risk of accumulation of hexose phosphates. The green line indicates the concentration of Fru-1,6-BP in the original model of glycolysis (17.2 mM, panel C, model A). Glce in this simulation is 25 mM. (E) Time course simulation of model D at 25 mM Glce and various values for the Vmax,ATP:ADP antiporter parameter. Plotted is the concentration of glycosomal phosphates (ΣP similar as in Figure 2, moiety 5 in Table 2). ATP:ADP antiporter activity values below 1 nmol·min−1·mg protein−1 result in depletion of glycosomal phosphates (cf. Figure 2). kTOX = 2 µl·min−1·mg protein−1 in all models. Solid lines indicate medians, shaded areas and error bars show interquartile ranges, as derived from the uncertainty modeling.
Figure 4.
Simulations of oxidative stress in model C.
(A) The steady state flux through the cytosolic pentose phosphate pathway in model C as a function of the oxidative stress by varying the kinetic constant kTOX. (B) Fluxes through the cytosolic PPP enzymes as a function of time upon sudden oxidative stress. During the whole time-course, kTOX = 2 µl·min−1 · mg protein−1. The system is removed from steady state at t = 0, by setting 99% of the NAD(P)H and trypanothione pools to the oxidized form. Shown is the relaxation of the cytosolic PPP fluxes. Solid lines indicate medians, shaded areas show interquartile ranges. Near identical results were obtained for model D (Figure S5).
Figure 5.
Simulations of 6PGDH inhibition and 6-PG accumulation.
(A–B) The effects of inhibition of 6PGDH on 6-PG concentrations and metabolic fluxes were simulated by reducing Vmax,6PGDH in model C and D at high oxidative stress (kTOX = 200 µl·min−1·mg protein−1). Simulations at low oxidative stress (kTOX = 2 µl·min−1·mg protein−1) are shown in Figure S6. ATP production flux as steady-state flux through PFK is indicated in red, while trypanothione reductase steady-state flux is indicated in yellow, both plotted on the left y-axis. Steady-state concentration of cytosolic (blue) and glycosomal (green) 6-phosphogluconate are plotted on the right y-axis. Shaded areas indicate interquartile ranges. (C) Steady-state flux through glycolysis as a function of the glycosomal 6-PG concentration in model A. A glycosomal 6-PG concentration of around 500 mM reduces the glycolytic flux by 50%.
Figure 6.
(A) Effect of 6PGDH ablation on the growth rate. A non-induced 6PGDHRNAi culture, grown in glucose-containing HMI-9 was split at 0 h, +tet is induced with tetracycline, while −tet is the non-induced control. (B) Specific activities of 6PGDH in induced and control 6PGDHRNAi parasites. (C) Western blot showing predominant co-localization of 6PGDH with the glycosomal marker aldolase (fraction S), while a faint band can also be observed in the cytosolic fraction P with the marker enolase. (D) Cell densities during growth on different substrates. At t = 0 h, a 6PGDHRNAi culture grown on glucose was split at 1·105 ml−1 to HMI-9 with either glucose or fructose, and in the absence and presence of tetracycline. Plotted cell densities are cumulative, as −tet cultures were split at 48 h to 1·105 ml−1. A higher starting cell density was used to allow the parasites to adapt to the change in carbon source. Growth on fructose is slower than on glucose, but is unable to rescue the induced cultures.