Growth Autonomy 2

Question 1

What are the members of the monomeric G proteins superfamily - Ras?

Answer 1

Rac

Rho

Rab

Arf

Question 2

Example what each of these are in the biochemical, structural homology of HRas, Kras 4a & b and, NRas.

G domain

C terminla domain

CAAX region

Answer 2

• G domain: allows Ras proteins to act as monomeric gene proteins and contains death binding regions and various others so very conserved
• C terminal domain: very little conserved, lots divergence, equally proficient at driving cell cycle
• CAAX motif: localises Ras to membrane where it needs to be for its funcionality

Question 3

Outline the Ras signalling cycle.

3 marks

Answer 3

• Ras when bound to GDP is inactive when bound to GTP in active conformation. Will switch itself off by hydrolysing 2GTP so back to inactive shape
• To cycle between conformations needs accessory proteins – guanine exchange factor (GEF) sos binds to ras and changes conformation to get rid of GTP and get GDP from cytosol
• Needs help with hydrolysis as have poor intrinsic GTPASE activity – hydrolysis is induced by GAP

Question 4

Outline the molecular basis of the G protein conformational change

3 marks

Answer 4

• Binding of GTP crucial to conformational change
• On GDP - Two regions known as switch regions in protein so cant bind to any effector molecules – GDP
• Third phosphsate interacts with two highly conserved amino acids in switch region – causing massive conformational change in gene protein allowing it to interact with its effector e.g. pi3 kinase or raf

Question 5

Which isoform of Ras is the most mutated and what does said mutation do?

Answer 5

• KRas mutations have the highest incidence and they render Ras constituitively active by preventing GTP hydrolysis
• Common point mutations alter AA residues at positions 12, 13 or 61

Question 6

What are the hotspot mutations in Ras?

2 marks

Answer 6

AA 12 or 13 - point mutation here causes massive effect on activity of ras

Question 7

How do mutations in Ras render it constitutively active?

Answer 7

By preventing GTP hydrolysis

Question 8

How can Ras be inappropriately activated?

Answer 8

GAP deletion or mutation

GAP deletion:

• common GAP associated with RAS is neurofirbin

Mutation:

• mutations in neurofribrim gene NF1 cause dysregulated cell growth

Question 9

Why is membrane localisation of ras important for its activity and how is it achieved?

4 marks

Answer 9

• Important to be able to receive signals
• Achieved by multi-step post-translational modifications
• First step: addition of isoprenoid lipid (farnesyl isoprenoid) to CAAX by farnesyl transferase
• Specified by variable region and CAAX motif

Question 10

How does the post-translation modifications of Ras proteins take place?

7 marks

Answer 10

• 1st step occurs in cytosol and using substrate FFP enzyme farneyl transferasewill transfer farneysl onto csyteine of CAAX motif
• Using lipid farnesyl Ras will translocate and acnhor to outside of ER membrane
• Once anchored becomes substrate for enzyme Race which cleaves off 33 AA
• ICMT then methylesterifies the carboxyl group [on membrane as both race and ICMT exclusively located on membrane]
• Palmotiyl lipid group added by palmotyiltransferase onto Ras
• Using farneysl and palmitoyl group, Ras will anchor onto membrane
• ABOVE ONLY TRUE FOR NRAS, HRAS, KRAS4A
• Not for Kras4B has lycine rich region in variable region - can sub for palmitoyl bit so Kras4b doesn’t have palmitoyl group

Question 11

What was seen in preclinical studies with farmesyl transferase inhbitors?

2 marks

Answer 11

• Significant potency as an ant-cancer drug for wide panel cancer cell lines
• Activity independent of Ras mutational status

Question 12

What was seen in in vivo models of farnesyl transferase inhibitors?

2 marks

Answer 12

• Activity in xenograft tumours regardless of Ras mutation
• Little toxicity when combined with toxic drug

Question 13

What was seen in clinical trials with FTIs?

1 marks

Answer 13

• Patients showed little to no response or ad some stability for very short period of time

Question 14

Why did patients react in certain way in the clinical trials with FTIs?

3 marks

Answer 14

• Kras and Nras still being prenylated and localised from cells
• Only Hras was actually farnesylated
• Kras and Nras can be alternatively prenylated through addition of geranylgeranyl 20-carbon isprenoid moiety by GGTase

Question 15

Why couldn’t an anti-cancer drug for Kras be used?

Answer 15

Drugs too toxic as knocking out too many proteins can’t be used in clinic

Question 16

How else can you target Ras that doesn’t involve knocking out Kras?

Answer 16

By targeting the substrates needed for prenylation

Question 17

What is one way you can target Ras prenylation - through the use of what known drugs?

3 marks

Answer 17

• Statins - used to reduce CVD
• Ras pathway makes cholesterol so can use Lovastatin to block 3-hydroxy-3methylglutaryl CoA reductase
• This reduces mevalonate products i.e. FPP and GGPP needed for prenylation

Question 18

How can bisphosphates be used to inhibit prenylation?
2 marks

Answer 18

• Inhibits isopentyl-PP siomerase and farneysl-PP synthase
• (used in osteoporosis)

Question 19

What is the Kras binding problem?

Answer 19

Kras GTP pocket inaccesible due to high affinity for GTP as GTP binds to active KRAS

Question 20

What is the KRAS G12C opportunity?

4 marks

Answer 20

• Found inhibtior that can bind to the cysteine of Krasg12c in its inactie form
• Binding opens switch II pocket
• Inhibitor covalently binds to cysteine and induces switch II pocket
• Kras G12C irreversibly locked in inactive state

Question 21

In order to give Ras an inhibitor what needs to be known?

Answer 21

If a B-Raf mutation is present as that’s where activation is

Question 22

Which Raf kinases is the key target in drug therapy and why?

4 marks

Answer 22

• B-raf
• Found in 40-60% cutaneous melanomas
• Inhibited by small molecule kinase inhibitors
• Has substitutions of aline with glutamic acid at position 600
• Mutation allows Raf to act as fully active monomer and doesn’t need to bind to Ras for permanent downstream signalling

Question 23

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