32
1 Bi 1 “Drugs and the Brain” Lecture 21 Thursday, May 11, 2006 Cystic Fibrosis: A Genetic Disease Today’s Bi 1 teaser: a best-selling biotech product. There’s a hint in the lecture hall !
1
Bi 1 “Drugs and the Brain”
Lecture 21
Thursday, May 11, 2006Cystic Fibrosis: A Genetic Disease
Today’s Bi 1 teaser: a best-selling biotech product.
There’s a hint in the lecture hall !
2
1. Clinical description
2. Genetics
3. Gene structure
4. CFTR as a protein
5. Physiology of CFTR
6. What’s wrong with F508?
7. The cholera connection
8. Selective advantage of CF?
9. Therapeutic approaches:Incremental approachesGene therapy
3
gall bladder
common bile duct
cystic ductbecomesfibrotic(invaded by fibrous tissue)
1. Clinical Picture of cystic fibrosis
Skin is salty (usually the first sign).
Thick, sticky mucous covers the respiratory tractand digestive tract.
Respiratory infections.
Until recently, patients rarely survived childhood.
Males infertile
small intestine
4
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).
CF
CF
CF phenotype
homozygousCF
CF
WT
normal phenotype
heterozygous“carrier”
CF
WT
normal phenotype
heterozygous“carrier”
WT
WT
normal phenotype
homozygousnormal
heterozygousmutant mother
“carrier”
CF
WT
normal phenotype
heterozygousmutant father
“carrier”
CF
WT
normal phenotype
1 marriage in 400 involves 2 heterozygotes
noncomplementation:1 birth in 1600 has CF
like Lecture 202. CF Genetics
5
3. Gene structure (discovered in 1989)
Long arm of chromosome 7
(from Lecture 20)
6a,b 14a,b 17a,b
alternatively spliced exons
250 kb in length
27 exons, 1480 amino acids = 4440 nt coding region (only 2% of the gene!)
6
4. The Protein Encoded by the Cystic Fibrosis Gene: Cystic Fibrosis Transmembrane Conductance Regulator (CFTR)
A Cl- channel2 sets of 6 transmembrane domains
2 “ATP-binding cassettes” (thus “ABC transport protein”)
N
CR-domain
Cl- out
in
unique among channels and ABC transporters
ATP ATP
7
from Lecture 6
The Nobel Prize announcement for the patch clamp highlighted cystic fibrosis
http://www.nobel.se/medicine/laureates/1991/press.html
Press release for 1991 Nobel Prize in Physiology or Medicine:
8
Until 2002, we had only guesses about the atomic-scale structures of ABC transport proteins
R
Big Alberts 11-16© Garland Publishing
9
http://www.sciencemag.org/cgi/content/full/296/5570/1091
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
you must have Swiss-protein viewer
http://www.its.caltech.edu/~lester/Bi-1/abc-protein.pdb
The E. coli BtuCD Structure:
A Framework for ABC Transporter Architecture and Mechanism
10
cytosol
kinase
phosphorylated protein is an ion channel
cAMPCa2+
intracellularmessenger
receptor
tsqiG protein
enzymechannel effector
from Lectures 12, 14
11
Gs
Enzymes are Gq, Gs, and Gt protein effectors
cyclic AMP (cAMP)ATP
N
NN
N
NH2
O
OHO
HH
O
P-O
O
cyclic AMP (cAMP)
N
NN
N
NH2
O
OHOH
HHCH2
H
OP
O-
O
OP
O-
O
-O OP
O-
OMg2+
ATP
Enzyme“cyclase”
Mg2+
receptor
tsqiG protein
cAMPCa2+
intracellularmessenger
enzymechannel effector
from Lecture 12
12
Ca2+ or cAMP binds to kinase;this activates the kinase as an enzyme.
The enzyme catalyzes:
ATP to ADPTarget protein
Phosphate attached toa specific amino-acid side chainof the target protein
kinase
phosphorylatedprotein
is an ion channel
cAMPCa2+
intracellularmessenger
modified from from Lecture 15
13
serine
tyrosine
NH
CHC
CH2
O
O
NH
CHC
CH2
O
O
-O OP
O
O
-O OP
O
O
NH
CHC
CH2
O
O
NH
CHC
CH2
O
O
OH
OH
kinase
phosphatase
otherkinases
otherphosphatases
kinase
phosphorylatedprotein
is an ion channel
cAMPCa2+
intracellularmessenger
from Lecture 15
14
N
C
R-domain
out
in
ATPATP
cAMP-activatedprotein kinase
Expression in frog eggs shows that . . .
CFTR is a Cl- channel activated by phosphorylation and by ATP breakdown
15
N
C
Cl- out
in
-O
OP
O
O
-O
OP
O
O-O
OP
O
O
O-
OP
O
OO-
OP
O
O
R-domain
ADP ADP
Expression in frog eggs shows that . . .
CFTR is a Cl- channel activated by phosphorylation and by ATP breakdown
16
Excised “inside-out” patch allows access to the inside surface of the membrane
no additions no channel openings
CFTR is a Cl- channel activated by phosphorylation and by ATP breakdown
Like Lecture 12
cAMP-activatedprotein kinase
+ATP
closed
open
cAMP-activatedprotein kinase
+ATP
Cell expressing CFTR
17
cytosol
How fast?10 s
kinase
phosphorylated protein is an ion channel
cAMPCa2+
intracellularmessenger
receptor
tsqiG protein
enzymechannel effector
from Lectures 12, 15
18
Na+ Na+ 1. Na+ channels are usually open, but extensive Na+ flux requires a counterion.
Cl-
2. If CFTR is open, Cl- becomes the counterion. Therefore NaCl flows across the membrane.
Cl-
4. Result: isotonic NaCl solution flows from the blood to the lumen (or vice-versa)
3. Water then flows around the cells to maintain osmotic pressure.
water
the blood surface is usually quite permeable to ions
lumen“apical”surface
CFTR helps to control bulk water flow across epithelia
Epithelium: layer of cells closely bound to one another to form a continuous sheet.
19
1. coilThe osmotic gradient favors secretion of salt.“Primary” sweat is isotonic.
2. ducta. 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.
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.
20
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.
N
CR-domain
Cl- out
in
6. What’s wrong with F508?
21
WT CFTR
CFTR-F508
CFTR-F508 does not usually reach the cell membrane;
but under special conditions, CFTR-F508 does reach the cell membrane and functions correctly!
22
cytosol
kinase
phosphorylated protein is an ion channel
cAMPCa2+
intracellularmessenger
receptor
tsqiG protein
enzymechannel effector
7. Cholera toxin bypasses receptors and highjacks the Gs pathway
Cholera is a bacterial disease of the intestines. Cholera and related diseases produce a toxin that enters cells.
23GTP GDP + Pi
Effector: enzyme
outside
inside
Neurotransmitter or hormonebinds to receptor
activatesG protein
The G Protein-Coupled Receptor Pathway
Cholera toxin blocks the GTPase of the subunit of Gs. The result: Gs is permanently activated.
24
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.
25
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.
8. Selective advantage of the CF gene
26
Therefore we set 0)1(2 2 ppsp , and we find that 212 ppps . 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.
WT / WT WT / CF CF / CF
fraction of offspring before selection
incremental offspring after selection
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).
2)1( p )1(2 pp 2p
0 )1(2 psp 2p
Hardy-Weinberg law, 1908
27
The CF mutations--some 500 throughout the gene--are restricted primarily to northern European populations.
What is the explanation?
28
All I really need to know about lifeI 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
29
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
9. How close is a “cure” for CF?
30
1. Clinical description
2. Genetics
3. Gene structure
4. CFTR as a protein
5. Physiology of CFTR
6. What’s wrong with F508?
7. The cholera connection
8. Selective advantage of CF?
9. Therapeutic approaches:Incremental approachesGene therapy
31
Contributions to CF research have come from
bacteriavirusesfrogs and frog eggsrats micefruit fliesyeasthealthy peoplepatients
Merlin as a model for biological advances?
The Sword in the Stonehttp://www.amazon.com/exec/obidos/ASIN/0399225021/qid=958102821/sr=1-5/102-9056065-8068845
Newton as a model for scientific advances?
32
Bi 1 “Drugs and the Brain”
End of Lecture 21
