Lecture 8 - Molecular Basis of Cystic Fibrosis Flashcards Preview

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Flashcards in Lecture 8 - Molecular Basis of Cystic Fibrosis Deck (49):
1

On which chromosome is the CFTR gene?

Cr 7

2

Length of CFTR gene

70kB

3

Number of exons in CFTR gene

27

4

What is the CFTR protein?

A large, integral, glycosylated, membrane-spanning protein

5

Weight of CFTR protein

170kD

6

Is CFTR responsible for active transport?

No.
Despite using ATP, CFTR moves Cl- with concentration gradient

7

Which family does CFTR belong to?

ATP-Binding Cassette (ABC) superfamily of membrane transporters

8

How is CFTR channel regulated?

By cAMP-dependent phosphorylation

9

Which cells express CFTR?

Epithelial cells, normally on apical surface

10

What are the domains of the CFTR protein?
1)
2)
3)
4)
5)

1) Membrane-spanning domain 1
2) MSD 2
3) Nucleotide-binding domain 1
4) NBD 2
5) Regulatory domain

11

Function of membrane-spanning domains

Form pore through which Cl- move

12

Function of nucleotide-binding domains

Bind and hydrolyse ATD

13

Function of regulatory domain

Several sites that can be phosphorylated by cAMP-dependent kinases

EG: Protein kinase A

14

Examples of different direction of Cl- flow through CFTR

Lungs: Cl- flows out of cell

Sweat duct epithelial cells: Cl- flows into cells

15

How does CFTR interact with other proteins?

Largely through C-terminal, which is anchored to cytoskeleton

16

Example of protein with which CFTR interacts

ENaC sodium channel

17

Domain on C-terminal of CFTR which interacts with other proteins

TRL - Threonine - Arginine - Leucine

18

Normal function of CFTR in cell in lung
1)
2)

1) Cl- moves out of cell through CFTR
2) Na+, H2O move into cell down concentration gradient

19

Defective function of CFTR in cell in lung
1)
2)
3)

1) Cl- ions can't escape cell through CFTR
2) Buildup of Cl- in cell results in a greater concentration gradient.
3) ENaC is not inhibited by CFTR. Unregulated uptake of Na+ into cells, leading to water osmotically being absorbed into cells. Dehydration of ASL

20

Normal function of CFTR in a sweat duct
1)
2)

1) Cl-, Na+ and H2O enter cell down concentration gradient
2) Sweat is secreted, but ions are largely reabsorbed by cells

21

Defective function of CFTR in a sweat duct
1)
2)

1) Cl- can't enter cell, so Na+ ions don't either
2) Buildup of Cl- and Na+ ions in sweat, leading to abnormally salty sweat

22

Number of known mutations in CFTR

Over 1900

23

Where are most mutations found in CFTR?

Exons 4, 8, 14, 20

24

Most frequent mutation in CFTR

Missense

25

Class I CFTR defect

No protein production

26

Class II CFTR defect

Defective processing
(maturation, premature degradation)

Protein can't leave ER, Golgi

27

Class III CFTR defect

Defective regulation
(defective ATP binding, hydrolysis)

Channels don't open

28

Class IV CFTR defect

Defective or reduced opening of ion channel, ion conductance

Channels can open, but not much

29

Class V CFTR defect

Reduced protein production
(promoter or splicing abnormality)

30

Class VI CFTR defect

Accelerated turnover from cell surface

Quite a rare type of defect

31

Common mutations leading to a class I defect

Nonsense, missense, frameshift mutations

32

Do mutations in CFTR gene have to be pathogenic?

No.
Some are non-pathogenic, some have reduced penetrance

33

What could lead to a class VI defect?

Nonsense mutation placing a stop codon near the C-terminal

Protein instability at cell surface

34

What could lead to a class III defect?

Mutation in nucleotide-binding domain 2.
ATP can't be hydrolysed

Or mutation in R domain

35

What could lead to a class II defect?

Mutation in nuclear binding domain 1.
Defective cell processing

This is where F508del occurs.

36

What could lead to a class IV defect?

Mutation in membrane-spanning domain 1.
Cl- have more trouble moving through pore

37

What could lead to a class I defect?

Mutation in membrane-spanning domain 1.
Normal levels of mRNA, absent protein

38

What could lead to a class V defect?

Mutation in membrane-spanning domain 1.
Reduced number of transcripts

39

Most common mutation

F508del

40

Which class of defect results from F508del?

Class II
Protein is misfolded, retained in ER, degraded

41

Way to correct class I defects

Aminoglycosides
Allow 'read through' of mRNA

42

Way to correct class II defects

'Correctors' to improve protein processing

43

Way to correct class III defects

'Potentiators' to activate protein

44

Way to correct class IV defects

Flavinoid compounds to improve channel conductance
(channel more likely to be open)

45

Way to correct class V defects

Improve number of correctly-spliced mRNA molecules

46

How does F508del occur?
1)
2)
3)

1) C from end of isoleucine and TT from phenylalanine are deleted.

2) Last amino acid of phenylalanine codon (T) combined with first two amino acids of isoleucine codon (AT) leads to isoleucine codon.

3) 3-base, out of frame deletion, but only phenylalanine has been removed.

47

Frequency of F508del mutation

~50% CF sufferers homozygous for F508del
F508del accounts for ~75% of CFTR mutations in northern Europe

48

How was CFTR first discovered to be the protein involved in CF?

Positional cloning

49

Least common place in CFTR gene for a mutation to occur.

Promoter