Case Study #2: Cystic Fibrosis

Dr. Christine Bear, PhD studies

Cystic Fibrosis
Dr. Christine Bear, PhD studies
Cystic Fibrosis

Characterized the protein

encoded by the gene mutated
in patients with CF. (CFTR)
Researchers studying cystic fibrosis used fluorescent-
antibody staining to observe the location of normal CFTR in
cells of the lung epithelium of healthy people.

Teal = nuclei
Blue = - tubulin (cilia)
Red = CFTR

CFTR is seen by immunofluorescence in the membrane of

lung epithelial cells.
 How do we determine if a protein
is a channel and what kind?
1. Isolate and purify normal
protein
 How do we determine if a protein
is a channel and what kind?
2. Mix purified CFTR protein
4. Test for solute movement.
with phospholipids to make
liposomes.
When CFTR is present, Cl- ions move
across the membrane.
The amino acid sequence for one of the most common
CFTR variants found among CF patients is shown
below. This depicts only the piece of the protein that
contains the variation, specifically amino acids #484-
520.
H3N+hsgrisfcsqfswimpgtikeniifgvsydeyryrsvCOO
Normal: H3N+hsgrisfcsqfswimpgtikeniigvsydeyryrsvCOO
F508del:

1. What is the difference between the normal and

mutant protein?

2. What change in the DNA sequence would cause this

difference?
Where does the F508del protein localize?

Teal = nuclei
Normal Blue = -
CFTR tubulin (cilia)
Red = CFTR

F508de
l

Kreda et al.
2005
Why is mucus thick in CF patients?

With no Cl-
channel at the
membrane,
osmosis
drives water
in, producing
thick mucus
What are the consequences of thick
mucus in CF patients?
Researchers expressed normal CFTR in cultured cells. They treated the
cells with cycloheximide (a chemical that blocks translation) in the
presence or absence of a proteasome inhibitor drug. Following
treatment, the researchers quantified the amount of CFTR protein
remaining in the cells over four hours. The results of this experiment are
summarized in the graph below.

What happens to normal CFTR protein after cycloheximide

treatment in the presence or absence of proteasome inhibitor? Why
Next, the researchers repeated the experiment using F508del CFTR
protein.

What happens to F508del CFTR protein after cycloheximide

treatment in the presence or absence of proteasome inhibitor?
Why might this happen? What can you conclude from the results
of this experiment?
 The proteasome degrades most F508del CFTR
protein because it doesnt fold properly in the
RER.
CFTR: cystic fibrosis transmembrane conductance regulator

ER
vesicle bound
for golgi

Folded properly
In

good protein
endoplasmic reticulum
In bad protein

Folded improperly

ER
Proteasome
(degradation)

Cyr, DM (2005). Nat Struct Mol Biol. 12, 2.

 If we can get F508Del CFTR protein to
the membrane, does it still function?

Mix purified F508 CFTR protein

Test for Cl- conductance
with phospholipids to make
liposomes.
F508del CFTR membrane localization is
rescued by low temperature

Hypothesis: at low
temperature, the
folding process slows
down and some of
the protein folds well
enough to evade the
quality control
system in the RER!

Cholon et al.
2010
 The future of Cystic Fibrosis
treatment: corrector chemicals!