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Molecular cellular biology > GTPases > Flashcards

GTPases Flashcards

1
Q

what are small GTPases?

A
  • small 21kDa proteins
  • Ras superfamily - one of the largest groups of signalling proteins
  • change conformation upon activation and bind/activate downstream effectors
  • within these groups are many branches and subfamilies
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2
Q

what are the main subfamilies of the Ras-GTPase superfamily?

A

arf - membrane budding
rab - endosomal trafficking
rho - cytoskeleton and migration
ras - cell proliferation, oncogene

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3
Q

what is the structure of GTP?

A
  • made of guanine nucleotide found in DNA
  • guanine is bound to ribose, forming a guanosine nucleoside
  • the whole structure is completed by 3 phosphate groups: alpha, beta and gamma
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4
Q

how can GTP function as an energy source?

A
  • hydrolysis of GTP to GDP, by cleavage of the gamma phosphate, releases energy
  • this also causes a conformational change in the biochemical structure of the molecule
  • GTP is an unstable structure, so it is energetically favourable to hydrolyse GTP to GDP
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5
Q

when are GTPases active and inactive?

A

GTP-bound structure is active

GDP-bound structure is inactive

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6
Q

what is the role of the GTPase in signalling?

A

the GTPase hydrolyses GTP in order to cycle

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7
Q

why is cycling of GTPase important?

A

cycling of the GTPase is essential as the GTPase needs to stay dynamic to switch things on/off

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8
Q

what is the difference between ‘signalling-active’ and ‘hydrolysis-active’?

A

Signalling-active = bound to GTP
Hydrolysis-active = hydrolyse GTP to GDP to become inactive

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9
Q

what are the key structural properties of the GTPase?

A
  1. Phosphate binding by P-loop – crucial for nucleotide binding and controlling the shape of the GTPase as it coordinates the phosphates
  2. Mg2+ is essential for nucleotide binding
    - Divalent cation introduces strong positive charges to the molecule, so that the negatively charged phosphates bind tightly to the GTPase
  3. Switch regions bind effectors: switch 1 and switch 2
    - These bind to downstream effectors
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10
Q

how may GTPase activity be measured?

A
  • Effector binding is used to measure GTPase activity
  • Effector proteins are precisely matched to the P-loops, so can distinguish between the active and inactive GTPase
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11
Q

what are the 2 catalytic ways in which GTPase can be hydrolysed?

A
  1. Positioning of attacking water – catalytic glutamine
    - Water attacks bond between the beta and gamma phosphate
    - The GTPase glutamine positions the water in the best position to cause hydrolysis
  2. Counteracting of negative charge at phosphates to break the bond
    - P-loop (12GxxGKT17) contributes positive charge through hydrogen bonds and lysine, so neutralizes the negative charge and lowers the energy barrier to allow hydrolysis
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12
Q

what happens if the glutamine in the GTPase is mutated?

A
  • Q61L catalytic mutant – keeps the GTPase on at all times as it is constitutively bound to GTP which cannot be hydrolysed
  • G12V pushes Q61 out of position and disturbs P loop
  • Conflicts with transition state geometry, but fine in ground state
  • Mutations of the GTPase structure reduce hydrolysis 10-fold
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13
Q

how are GTPases such as Rac cyclically regulated?

A
  • Rac1-GDP can become active by replacing the GDP with GTP via GEF (guanine nucleotide exchange factor)
  • Rac1-GTP can then bind to downstream effectors and trigger a signalling pathway
  • GTPase hydrolysis of GTP to GDP is accelerated by GAP (GTPase Activating Protein) 2000-105 fold- the Rac1 is now bound to GDP and inactivate again
  • Rac1-GDP can be mopped up by GDI (Guanine nucleotide dissociation inhibitor) and sequestered in the cytosol, so the GTPase remains switched off
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14
Q

what is the role of the guanine nucleotide exchange factor (GEF)?

A

It accelerates exchange of GDP for GTP to turn on signalling:
- the GEF stabilises the transition from GDP to GTP and accelerates the exchange rate 10-107 fold
- it activates the GTPase activity so that it can effect downstream proteins

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15
Q

what is the role of the GTPase activating protein (GAP)?

A

These activate GTPase hydrolysis to turn off signalling:
- GTPase hydrolysis of GTP to GDP is accelerated by GAP (GTPase Activating Protein) 2000-105 fold
- the GTPase can then bind to GDP and become inactive

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16
Q

how do GAPs catalyse the hydrolysis of GTP to GDP?

A
  • Flexibility of Arg85 helps – it has a strong positive charge which counteracts the negative charge of the phosphates to destabilise the phosphate bounds and aiding hydrolysis
  • Arg85 inserts into active site to stabilise transition state and positions Q61 properly for nucleophilic attack
  • Water has restricted freedom to move, so there is a reduced entropy barrier enabling acceleration of hydrolysis
  • Stabilises position of water and magnesium for optimal reaction
17
Q

how does the GEF aid in exchange from GDP to GTP?

A

it stabilises the nucleotide-free and magnesium-free form of Rac
- Easier for GDP to fall off the GTPase and for GTP to bind

18
Q

what is an example of a mutation in Rac which affects GEFs?

A

T17N is a dominant negative mutant of Rac
- Thr 17 stabilises Mg2+
- T17N Mg2+-free so mops up GEFs and switch off GTPase of the cell

19
Q

what are the families of GEFs?

A
  • Dbl-homology domain GEFs
  • DOCK-family GEFs
  • Sec7 domain GEFs
20
Q

which GEFs lack specificity?

A

Vav1/2/3 are not specific to any GTPase, and assist in nucleotide exchange for all GTPases

21
Q

give an example of a GEF that is specific:

A

Tiam1: 9 residues from body and Switch 2 form contiguous contact
- Regulates Rac

22
Q

how do mutations show how specific GEFs can be?

A

Rac W56F mutation is Tiam1 insensitive but Itsn1 sensitive, so can bind to a different GEF
- A single residue substitution can change GTPase Rac to Cdc42, showing how specific they are

23
Q

what may trigger actin-based cell motility to occur?

A

chemical signals
e.g. stimulation of the cell with a growth factor will trigger the cell to move towards that growth factor

24
Q

what structures are formed during actin-based motility?

A

Filopodia spikes will form via activation of Cdc42

Lamellipodium sheet will form and protrude towards the signal to form adhesions, via activation of Rac1

Finally, formation of contractile actin stress fibres is triggered by RhoA
- adhesions at the front will dissemble to that the cell can be pulled forward

25
Q

what GTPase triggers filopodia spike formation?

A

Cdc42

26
Q

what GTPase triggers lamellipodia formation?

A

Rac1

27
Q

what GTPase triggers stress fibre formation?

A

RhoA

28
Q

what are the downstream effects of RhoA to trigger stress fibre formation?

A
  • GTP-RhoA will bind to and activate Rho-kinase
  • Rho-kinase will phosphorylate the Myosin Light Chain
  • MLC phosphorylation leads to the contraction of the actomyosin stress fibres
29
Q

how are the GTPase signals in actin-based motility antagonistic?

A
  • Cdc42 and Rac1 are protrusive signals – cell pushes forward at the front of the cell
  • RhoA is a contractile signal – pulls the rear end of the cell forward
30
Q

why is it important that the GTPase signals in actin-based motility are coordinated?

A

Coordination of these signals is important to ensure the cell is forming the right structure in the right sequence:
- During protrusion phase, there is high Rac levels and low RhoA levels
- During contractile phase there is high RhoA levels and low Rac levels

31
Q

what are the characteristics of migration in 2D?

A
  • Random
  • Flat
  • Fast
32
Q

what are the characteristics of migration in 3D?

A
  • Directional
  • Integrated within the matrix - can translocate more efficiently
  • Often slower
33
Q

what matrix do cells migrate in?

A
  • fibronectin matrix fibres within the ECM
34
Q

does increasing Rac1 activity make migration faster?

A

depends on when in the process its activity is increased:
- If Rac1 activity is increased, it needs to be increased at the right time in the protrusion phase
- Its activity cannot be increased during the contractile phase as it would interfere with RhoA

35
Q

why does migration require localised GTPase signals?

A

to ensure that the GTPases are at the correct pole of the migrating cell:
- Rac1 activation localised on the front end of the cell results in the cell moving in the correct direction
- If rac1 activity is dispersed all over the cell, the cell no longer has direction, so cannot follow along the fibres
- It may shunt backwards and forwards as they have no direction and cannot move purposefully
- If the migrating cells are skin fibroblasts, the fibroblasts will be unable to move efficiently to a wound, resulting in healing defect

Molecular cellular biology (13 decks)

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