Growth Factors + EGF

Question 1

What do growth factors do?

Answer 1

regulate cellular processes such as proliferation, differentiation and maturation

Question 2

What are cytokines

Answer 2

Cytokines are growth factors of blood cells:
hematopoiesis and immune cell differentiation

Question 3

Explain why the definition of a cytokine ands growth hormones don’t always fit

Answer 3

Cytokines can affect cells other than blood cells

Cytokines may lead to growth, but they might also
induce apoptosis (neutral definition)

Growth factors and cytokines are proteins, but we also know steroid growth hormones (don’t need proteins)

Question 4

Explain where EGF comes from/how it gets released

Answer 4

GPCR recieves saignal e.g. Ach
Causes conformational change in the GPCR that causes signal to be sent to a different membrane protein (either ADAM or MMP)
Causes cleavage of membrane proteins to release EGF
EGF then binds to an EGFR and causes the MAPK cascade

Question 5

explain simply the specificity of GH binding to GHR

Answer 5

Limited number of AAs recognised
If change them, change Kd and therefore output

Question 6

does the same signalling molecule do the same thing in every cell

Answer 6

NO

Question 7

What is the most widely used technique to study drug binding

Answer 7

SPR - Surface plasmin resonance

Question 8

Explain how SPR works

Answer 8

Uses bait and prey molecules

ligand binding (association) and then wash buffer over (dissociation)

takes minutes

Interested in rate ligand falls off

Bait is attached to gold film by a flexible tether

Incident loght shined and the reflected light angle is detected by a detector, angle changes depending in what is attched

Question 9

Explain immediate early gene expression in cell cycle control

Answer 9

MAP kinase cascade leads to immediate early gene expression

Make TFs, new proteins that control cell cycle: cyclins and CDKs controlled by Myc

Myc leads to a delayed response gene expression, important in call cycle regulation

After we have delated response, then G1-CDK can become active

Question 10

Explain the cyclin/cdk partner occurring at different cell cycle stages

Answer 10

G1: Cyclin D/Cdk4/6
G1/S: Cyclin E/Cdk2
S: Cyclin A/Cdk2/1
M: Cyclin B/Cdk1

Question 11

Explain kinase/cdk activation

Answer 11

Wee1 deactivates CDK by phosphorylation on tyrosine 15

CAK can activate CDK by phosphorylation on threonine 161 (even when this happens, still inactive as phosphorylation of Y15 still there, needs Cdc25)

Cdc25 activates CDK by dephosphorylation on tyrosine 15 (M checkpoint?)

Question 12

Explain how cyclin A/Cdk2 goes from low to high activity

Answer 12

Phosphorylation of Thr160 on the T loop of CDK2

Question 13

Explain steps of the cell cycle using CDKs

Answer 13

SEE PHOTO

Question 14

What is Nef

Answer 14

Viral protein

Inihibits progression from Ras to Raf

Found in HIV – promotes viral replication

Question 15

Explain p53 in the DNA damage checkpoint

Answer 15

normal conditions:
p53 is attached to Mdm2 and ubiquitinated and therefore degraded
When DNA damage occurs - kinase cascade leads to phosphorylation of p53 and releade of Mdm2 - p53 now active
P53 is a TF, and attaches to p21 gene

P21 is then transcribed - it regulates cdks, it is an inhibitor
When transcribed the p21 protein is produced - cdk inhibator (clamps over cyclin/cdk) and so stops cell cycle progression

Question 16

features of EGF

Answer 16

Epidermal growth factor

• about 40 residues:
• 3 disulphide bonds.
• two-stranded beta-sheet followed by a loop to a C-terminal short two-stranded sheet.
• Subdomains (between the conserved cysteines) vary in length

Question 17

What does cbEGF stand for

Answer 17

Calcium bindinhg EGF

Question 18

Features of cbEGF

Answer 18

Often as tandem repeat

Main building block for fibrillin

Question 19

Explain the domain organisation of Fibrillin

Answer 19

Ca2+ binding EGFs – about 37

Also other domains – TB domain

A few EGFs that are non-calcium binding

Hybrid domains (2)

at the N terminus theres a unique region

Also a proline rich region

Question 20

function of fibrillin

Answer 20

Forms microfibrils (8 fibrillins) - part of the ECM

Question 21

Diseases caused by fibrillin mutations

Answer 21

500 mutations - all associated with a class of diseases called Marfan synrdome

Mild - Tall + lanky
Severe - Cardiovascular problems, vision problems, lethal at development stage

Question 22

What to EMs show about microfibrils?

Answer 22

show elongated ‘beaded’ structures

Question 23

What techniques can/cant be used to see microfibril structure

Answer 23

EM (doesnt show much though)
NOT - CryoEM, Crystallisation as fibril is too complex in structure
only option is to ‘dissect and conquer’

Question 24

two microfibril models

Answer 24

1. antiparallel staggered conformation – fibrillins layed out partially overlapping generating a repetitive pattern with a 50nm register
2. “Jack knife” model – Fibrillin arranged in a jack knife conformation and then these are arranged on top of each other – there is growing evidence that this is the correct model
   -Generation of elongated structures from domain building blocks
   -domain junctions potential hinge regions

Question 25

Features of cbEGF-cbEGF junctions

Answer 25

1 residue linker, well conserved interface (discovered through sequence alignment)
Rigid elongated connection

Question 26

cbEGF-TB junction features

Answer 26

variable linker, interface not conserved

experimentally observed flexibility