Lecture 19 - Regulation of Cell Migration Flashcards
What are the 4 different small GTPase families ?
Rab - Endosomal trafficking
Arf - Membrane budding
Ras - Cell proliferation, oncogene
Rho - Migration/cytoskeleton
small 21 kDa proteins
One of the largest groupps of signalling proteins
How is a GTPase usually activated?
GTPase is activated and undergoes subtle coinformational changes when a ligand binds. This then activates downstream effectors
What is the structure of GTP and what happens when it is cleaved?
Guanine nucleotide bound to a ribose molecule to make Guanosine. The energy part is the alpha, beta, and gamma phosphates.
When gamma phosphate is clipped off it will release energy and the molecule will have less charge. It is now in a more unstable form.
What is the difference between signalling and hydrolysis active ?
Signalling active molecule will be bound to GTP but hydrolysis active will chop off phosphate and switch off the signalling ability.
Why is cycling essential?
The ability of a GTPase to cycle its bound nucleotide is important for the cyclic nature of migration.
Arf6 is a cycling essential, ‘active Arf6’ is inhibitory
What is the P-loop?
Phosphate binding loop. These phosphates are coordinated by P loop, which controls shape of GTPase.
What does magnesium do in the GTPase molecule?
Essential for nucleotide binding
What do the switch regions do ?
Switch region - they will bind to the downstream effectors. These conformational changes are very subtle allowing it to bind to an effector.
How would we detect an active GTPase?
Effector binding – because GTPase is not a kinase so can’t use phosphorylation. Cannot use an antibody because the conformational change is so small the antibody will bind regardless of whether is active enough. We lack the ability to create a molecule that can do this but naturally effectors can that is why we use an effector
How does GTPase work as a catalyst of GTP hydrolysis using water?
The bound nucleotide is energetically favourable to be cleaved so there is an energy barrier that hydrolysis. GTPases act as an enzyme and position H2O to attack the bond between beta and gamma phosphate and hydrolyse the bond. The GTPase will position attacking water in the best place they can break the bond.
How does GTPase work as a catalyst of GTP hydrolysis using water?
The bound nucleotide is energetically favourable to be cleaved so there is an energy barrier that hydrolysis. GTPases act as an enzyme and position H2O to attack the bond between beta and gamma phosphate and hydrolyse the bond. The GTPase will position attacking water in the best place they can break the bond.
If we wanted to have an always GTPase always switched on always in the GTPase bound form how would we do that ?
We would substitute the glutamine. This will cause it to not be active.
Or substitute glycine 12 in the P loop will push Q61 out of position.
Counteracting negative charge on phosphates because the phosphate bond must be broken so again p loop by contributing positive charge will neutralize some negative charge and lower that energy barrier.
How can we switch a GTPase on and off?
Rac1GDP. We need to remove GDP to replace it with GTP. This is an unstable structure so Guanine Nucleotide Exchange Factor GEFs will stabilize the transition state so we can exchange bound nucleotides and accelerate the exchange rate. GEF is crucial for switching on.
Rac1GTP will bind to downstream effectors and initiate a signalling pathway.
GTPase will hydrolyse bound nucleotide. GTPase Activating Protein GAP proteins will assist in the hydrolysis of bound nucleotides.
Guanine Nucleotide Dissociation Inhibitors GDI are dissociation inhibitors that will bind to this form of GTPase old it in cytosol taking it out of the equation and therefore turning it off.
How can we switch a GTPase on and off?
Rac1GDP. We need to remove GDP to replace it with GTP. This is an unstable structure so Guanine Nucleotide Exchange Factor GEFs will stabilize the transition state so we can exchange bound nucleotides and accelerate the exchange rate. GEF is crucial for switching on.
Rac1GTP will bind to downstream effectors and initiate a signalling pathway.
GTPase will hydrolyse bound nucleotide. GTPase Activating Protein GAP proteins will assist in the hydrolysis of bound nucleotides.
Guanine Nucleotide Dissociation Inhibitors GDI are dissociation inhibitors that will bind to this form of GTPase old it in cytosol taking it out of the equation and therefore turning it off.
In GAPs what does Arg85 do ?
The flexibility of Arg helps, Arg inserts into active site to stabilise transition state and positions Q61 properly
Mechanism conserved, structure completely different.
