Lecture 5 glycolysis

Question 1

Why is glycolysis a hot topic?

Answer 1

• Glycolysis is a hot topic due to Warburg effect in cancer

Question 2

What is Warburg effect in cancer cells?

Answer 2

In oncology, the Warburg effect is a form of modified cellular metabolism found in cancer cells, which tend to favor the glycolysis over oxidative phosphorylation
(The Warburg Effect is defined as an increase in the rate of glucose uptake and preferential production of lactate, even in the presence of oxygen) So, cancer cells plunder more glucose from microenvironment and secrete more lactic acid to meet requirement of energy and material metabolism.

Question 3

What happens in the upper- and lower part of the glycolysis in yeast?

Answer 3

• Upper part (glycolysis) where you yield DHAP and GAP. Belangrijk: je gebruikt 2x ATP (‘als fosfaatdonor’) om op te starten (en fructose 1,6-bifosfaat te maken: FBP).
• Deze FBP hak je in 2en
• Lower part (glycolysis) where you yield ethanol: stop je de gehakte FBP in en haal je 2x 2 ATP uit. Netto opbrengst = 2 ATP. Wij mensen maken er melkzuur van, gist maakt er ethanol van

o The product of tps1 inhibits hk and stimulates lower part by providing phosphate
- Lower part needs phosphate that is usually obtained by the upper part

Question 4

Wat is trehalose?

Answer 4

• Trehalose is essential component of many vital physiological processes: heat and osmo stress
  o Is made when yeast is in stress in the upper glycolysis

Question 5

What happens with yeast & ATP, G6P (wordt trehalose van gemaakt) and F16bP when knocking down tps1?

Answer 5

• Conclusion: without a functional Tps1, cells cannot grow on glucose.
  What happens is: ATP remains constant, G6P remains constant, but F16bP, the particle between the upper and lower glycolysis, is accumulating. So, there must be a disbalance between these two parts.

Question 6

o Why can F16bp accumulate, why is there no feedback inhibition?

Answer 6

One of the reasons: because the enzyme producing F16bp (PFK) is insensitive for its product (F16bP).

Question 7

Wat gebeurt er wanneer PFK overexpressed wordt? Leg uit waarom

Answer 7

PFK: wordt negatief geremd door een hoge concentratie ATP (en dus laag AMP) PFK vertelt de glycolyse dat er meer ATP gemaakt moet worden. ATP gaat beetje omhoog, maar remt weer PFK.

Bij overexpressie PFK blijft glycolytische flux gelijk en gaat de hoeveelheid ATP pici omhoog.

Question 8

What happens if you increase AMP?

Answer 8

o What happens if you increase AMP: glycolytic flux goes up. High AMP = low ATP.

Question 8

What happens if you increase AMP?

Answer 8

o What happens if you increase AMP: glycolytic flux goes up. High AMP = low ATP.

Question 9

T6P, stofje in de trehalose cyclus, wordt gemaakt door tps1 en remt hk om te zorgen dat het bovenste gedeelte niet te hard van stapel loopt. Wrm maakt het qua remming van hk niet uit dat F16bp accumuleert?

Answer 9

Bleek foutief, want toevoegen van een random hk zorgt nog steeds voor accumulatie van F16bp.

Question 10

Waarom is fosfaat belangrijk in dit verhaal?

Answer 10

• The importance of phosphate: accumulation of phosphorylated intermediates requires phosphate uptake or mobilization (accumulation of F16bp) Vacuole in yeast that can provide phosphate

Question 11

How does the imbalanced state go back to the equilibrium state? How can you have two equilibrium states?

Answer 11

through adding phosphate. 2 eq states door positive feedback. Other wise negative feedback: only one state

Question 12

What does bistability mean?

Answer 12

Bistability: In a dynamical system, bistability means the system has two stable equilibrium states. Something that is bistable can be resting in either of two states.

Question 13

What is a simple experiment (or 2) to show bistability?

Answer 13

Bistabiliteit getest door: petrischaal uitplaten. WT groeit altijd op Glu/Gal. Tps1 mutant groeien er een paar op glucose, als je maar genoeg cellen gebruikt. Toen zeiden mensen: Ja dat zijn mutanten van mutanten. Volgende experiment: Als het door een mutatie komt, kunnen die glucose-positieve mutanten blijven groeien erop. Die mutanten strijk je op galactose (‘geheugen wissen’) en daarna weer op glucose. Dan zie je weer dat maar 1 op de zoveel groeien. Toont aan dat het niet genetisch is maar fenotypisch

Question 14

What types of decisions does bistability make?

Answer 14

• Bistability is everywhere in biology
  o To grow or not to grow
  o To sporulate or not to sporulate
  o To induce a pathway or not
  o To differentiate into one cell type or another
• Multiple states result in cell going into either one direction or other direction depending on trigger

Question 15

What is the conclusion of the (2) experiments?

Answer 15

 Conclusion: environment decides growth, not genetics

Question 16

Wat gebeurt er bij het toevoegen van alcohol/mierenzuur?

Answer 16

subpopulatie verkleint (?). Meer NADH

Question 17

Hoe kan je het metabolisme in single-cells aantonen?

Answer 17

• Assessing single-cell metabolism via pH: less ATP = lower pH = when giving glucose
  o Phenotypes pH of the yeast is different when they should be the same since they are from the exact same colony
  o Again proves that yeast adapts to environment
• Dynamic single-cell pH data
  o Creation of two subpopulations

Question 18

What is the lag phase and what does it reflect? Why is it a bad marker to determine if something will grow?

Answer 18

• Lag phase: phase where there is no growth yet.
• Lag phases reflect size of subpopulations.
• Playing with the subpopulations, low lag phase means a small subpopulation
• Therefore, Lag phase is a bad factor to determine if something will grow. Yeast would normally grow within minutes, a long lag phase merely means that the yeast is in the imbalanced state and not able to grow.

Question 19

Why is missing tsp1 causing a start up problem? Explain the model

Answer 19

Problem: phosphate is decreasing because you’re pumping in phosphate in FBP

o Drop in phosphate prevents from going back to the steady state but only stimulates the lower part

o 4 ATP is created when only 2 ATP is needed resulting in upper part being more active, this positive feedback maintains imbalance

o Tps1 is only needed for the first few minutes to push it into the good state : to avoid the drop in phosphate

o By adding phosphate in imbalanced state, you slow down G6P because of tsp1 and extra phosphate is created

Question 20

What was done during the dynamic labelling studies ?

Answer 20

• To measure the 13 C glucose concentration at different time points and determine how quickly glucose is used by the tsp1 cycle