Today’s Bi 1 teaser:
a best-selling biotech product.
There’s a hint in the lecture hall !
Bi 1
“Drugs and the Brain”
Lecture 21
Thursday, May 11, 2006
Cystic Fibrosis: A Genetic Disease
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1. Clinical description
2. Genetics
3. Gene structure
4. CFTR as a protein
5. Physiology of CFTR
6. What’s wrong with DF508?
7. The cholera connection
8. Selective advantage of CF?
9. Therapeutic approaches:
Incremental approaches
Gene therapy
2
1. Clinical Picture of cystic fibrosis
gall bladder
Skin is salty (usually the first sign).
cystic duct
becomes
fibrotic
(invaded by
fibrous tissue)
common
bile duct
Thick, sticky mucous covers the
respiratory tract
and digestive tract.
Respiratory infections.
Until recently, patients rarely survived
childhood.
Males infertile
small
intestine
3
like Lecture 20
2. CF Genetics
CF is the most common autosomal recessive disease in northwestern European populations.
The chromosome frequency is ~ 1/40:
One person in 20 carries a defective CF gene and is a heterozygote (WT / defective CF).
heterozygous
mutant mother
“carrier”
1 marriage in
400 involves 2
heterozygotes
heterozygous
mutant father
“carrier”
CF
CF
WT
WT
normal phenotype
normal phenotype
noncomplementation:
1 birth in 1600 has CF
homozygous
CF
heterozygous
“carrier”
heterozygous
“carrier”
homozygous
normal
CF
CF
CF
WT
CF
WT
WT
WT
CF phenotype
normal phenotype
normal phenotype
normal phenotype4
(from Lecture 20)
3. Gene structure (discovered in 1989)
Long arm of chromosome 7
alternatively spliced exons
6a,b
14a,b
17a,b
250 kb in length
27 exons, 1480 amino acids = 4440 nt coding region (only 2% of the gene!)
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4. The Protein Encoded by the Cystic Fibrosis Gene:
Cystic Fibrosis Transmembrane Conductance Regulator (CFTR)
A Cl- channel
2 sets of 6 transmembrane domains
2 “ATP-binding cassettes” (thus “ABC transport protein”)
Cl-
out
in
N
ATP
ATP
R-domain
unique among channels
and ABC transporters
C
6
from Lecture 6
The Nobel Prize announcement for the patch clamp highlighted
cystic fibrosis
Press release for 1991 Nobel Prize in Physiology or Medicine:
http://www.nobel.se/medicine/laureates/1991/press.html
7
Until 2002, we had only guesses about the atomic-scale structures
of ABC transport proteins
R
Big Alberts 11-16
© Garland Publishing
8
The E. coli BtuCD Structure:
A Framework for ABC Transporter Architecture and Mechanism
Kaspar P. Locher,* Allen T. Lee, Douglas C. Rees*
Division of Chemistry and Chemical Engineering,
California Institute of Technology, Pasadena, CA 91125, USA
Science 296, p.1091 (2002)
you must be “functionally” on campus
http://www.sciencemag.org/cgi/content/full/296/5570/1091
you must have Swiss-protein viewer
http://www.its.caltech.edu/~lester/Bi-1/abc-protein.pdb
9
from Lectures 12, 14
receptor
G protein
i q s t
effector
channel enzyme
intracellular
messenger
Ca2+ cAMP
cytosol
kinase
phosphorylated protein
is an ion channel
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receptor
from Lecture 12
Enzymes are Gq, Gs, and Gt protein effectors
G protein
i q s t
effector
channel enzyme
intracellular
messenger
Ca2+ cAMP
ATP
2+
Mg2+
Mg
NH 2
N
N
O
O
O
-O P O P O P O CH 2 O
H
H
OOOH
OH OH
ATP
cyclic AMP (cAMP)
Gs
NH 2
N
N
N
N
N
Enzyme
“cyclase”
O
O
O
P
-O
O
N
H
H
OH
cyclic AMP (cAMP)
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modified from from Lecture 15
intracellular
messenger
Ca2+ cAMP
kinase
Ca2+ or cAMP binds to kinase;
this activates the kinase as an enzyme.
The enzyme catalyzes:
ATP to ADP
Target protein
phosphorylated
protein
is an ion channel
Phosphate attached to
a specific amino-acid side chain
of the target protein
12
from Lecture 15
intracellular
messenger
Ca2+ cAMP
kinase
serine
O
N CHC O
H
CH 2
kinase
OH
phosphatase
tyrosine
O
N CHC O
H
CH 2
other
kinases
O
N CHC O
H
CH 2
O
-O P O
O
phosphorylated
protein
is an ion channel
O
N CHC O
H
CH 2
other
phosphatases
OH
O
-O P O
O
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Expression in frog eggs shows that . . .
CFTR is a Cl- channel activated by phosphorylation and by ATP breakdown
out
in
R-domain
N
cAMP-activated
protein kinase
ATP
ATP
C
14
Expression in frog eggs shows that . . .
CFTR is a Cl- channel activated by phosphorylation and by ATP breakdown
Cl-
out
in
N
ADP
OP
O
O
O
O
O
P
O
O-
ADP
R-domain
-O
O
P
O
O
O
P
-O O
C
O
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Like Lecture 12
CFTR is a Cl- channel activated by phosphorylation and by ATP breakdown
Excised “inside-out”
patch allows access to
the inside surface of the
membrane
cAMP-activated
protein kinase
+ATP
Cell expressing CFTR
no channel openings
no additions
closed
cAMP-activated
protein kinase
+ATP
open
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from Lectures 12, 15
receptor
G protein
i q s t
effector
channel enzyme
intracellular
messenger
Ca2+ cAMP
cytosol
kinase
phosphorylated protein
is an ion channel
How fast?
10 s
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CFTR helps to control bulk water flow across epithelia
Epithelium: layer of cells closely bound to one another
to form a continuous sheet.
lumen
“apical”
surface
Cl-
Na+ water
Cl-
Na+
1. Na+ channels are
usually open, but
extensive Na+ flux
requires a counterion.
2. If CFTR is open, Clbecomes the
counterion. Therefore
NaCl flows across the
membrane.
3. Water then flows
around the cells to
maintain osmotic
pressure.
the blood surface is usually quite permeable to ions
4. Result: isotonic NaCl
solution flows from the
blood to the lumen (or
vice-versa)
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sweat gland
Horse can’t dilute their sweat.
This explains how humans can run
marathons, but horses can’t.
This explains how CF patients have
salty sweat.
2. duct
a. A Na+ pump in the blood-facing membrane keeps
the intracellular Na+ low.
b. This attracts Na+ and Cl- out of the lumen when
CFTR is activated.
c. But the tight junctions don’t allow water flow.
d. Result: the sweat is depleted of salt.
1. coil
The osmotic gradient favors secretion of salt.
“Primary” sweat is isotonic.
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6. What’s wrong with DF508?
Cl-
out
in
N
R-domain
The most common mutation (70% of mutants):
a single-codon (3-nt) deletion at position 508
in ATP-binding cassette #1.
A phenylalanine (F) is deleted.
C
20
CFTR-DF508 does not usually reach the cell membrane;
WT CFTR
CFTR-DF508
but under special conditions, CFTR-DF508 does reach the cell membrane
and functions correctly!
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7. Cholera toxin bypasses receptors and highjacks the Gs pathway
Cholera is a bacterial disease of the intestines.
receptor
G protein
i q s t
Cholera and related diseases produce a toxin
that enters cells.
effector
channel enzyme
intracellular
messenger
Ca2+ cAMP
cytosol
kinase
phosphorylated protein
is an ion channel
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The G Protein-Coupled Receptor Pathway
Neurotransmitter or hormone
binds to receptor
activates
G protein
Effector:
enzyme
outside
inside
b g
a
GTP
a
GDP + Pi
b g
Cholera toxin blocks the
GTPase of the a subunit of Gs.
The result: Gs is permanently
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activated.
Cholera patients lose salt and water through their intestines because CFTR is
constantly open.
This has caused death during past cholera epidemics.
Modern cholera therapy began (~ 1965) when physicians realized that they could
force the fluid/salt secretion pathway to function in reverse by feeding patients large
quantities of isotonic solutions. This therapy is required for a few days, until the
patient can mount an effective defense against the cholera bacteria.
The World Health Organization has developed a special “ideal” isotonic solution of
NaCl, dextrose, and other inexpensive small molecules.
However, if the WHO solution is not available, most experts recommend . . .
Isotonic popular drinks.
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8. Selective advantage of the CF gene
CF is so destructive in the homozygote that many people have postulated a
selective advantage for the heterozygote.
This advantage would balance the lethality of the the homozygote and would
explain how the CF mutations have not been eliminated by natural selection.
CF may protect against cholera and related diseases.
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Formal analysis of “heterozygote advantage”:
Let s = the increased fraction of offspring that are produced by the CF heterozygote;
but 0 = the number of offspring produced by the CF homozygote.
p = the fraction of the population with CF chromosomes (~ 0.05)
( p << 1; it’s hard to imagine a mechanism that would confer a great selective
advantage to the heterozygote while remaining lethal for the homozygote).
fraction of offspring
before selection
incremental offspring
after selection
WT / WT
WT / CF
CF / CF
(1  p) 2
2 p (1  p )
p
0
2sp (1  p)
 p2
2
HardyWeinberg
law, 1908
Therefore we set 2sp(1  p)  p 2  0 , and we find that s  p 21  p   p 2 .
Thus the steady-state proportion of CF heterozygotes equals twice the “advantage” enjoyed
by heterozygotes.
We may suggest that at some point in the past, roughly 2.5% more CF heterozygotes lived
to reproductive age than did WT homozygotes.
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The CF mutations--some 500 throughout the gene--are restricted
primarily to northern European populations.
What is the explanation?
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All I really need to know about life
I learned in Bi 1
1. If you want a job done right, get a protein
2. Electrical circuits explain many processes
3. Most processes follow an exponential time course
4. Most processes end with a Gaussian distribution
5. Optics can show lots of details
6. Some drugs produce quasi-permanent changes in gene activation
7. Some diseases are inherited
8. Osmosis explains many processes
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9. How close is a “cure” for CF?
a. Enzyme replacement tablets for the digestive enzymes that do not reach the
digestive tract.
b. DNAse and other tactics to minimize the bacterial infections of the airways.
c. Gene therapy to replace CFTR.
Goal: a virus should deliver (your favorite gene), and only (your favorite gene) into
the target cell, without
(1) monopolizing host protein synthesis
(2) replicating uncontrollably
(3) inducing immunological reactions
Adenovirus (a cold virus) seems most promising.
(1) 35 kb, a manageable size
(2) enters a variety of cells, especially in the respiratory tract
(3) expresses genes even in nondividing cells
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1. Clinical description
2. Genetics
3. Gene structure
4. CFTR as a protein
5. Physiology of CFTR
6. What’s wrong with DF508?
7. The cholera connection
8. Selective advantage of CF?
9. Therapeutic approaches:
Incremental approaches
Gene therapy
30
Contributions to CF research have come from
bacteria
viruses
frogs and frog eggs
rats
mice
fruit flies
yeast
healthy people
patients
Newton as a model for scientific advances?
The Sword in the Stone
http://www.amazon.com/exec/obidos/ASIN/0399225021/qid=958102821/sr=1-5/102-9056065-8068845
Merlin as a model for biological advances?
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Bi 1
“Drugs and the Brain”
End of Lecture 21
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