Download CFTR and Kalydeco Presentation

Genes Genomes and Personalized Medicine
CFTR … and Kalydeco
Cystic Fibrosis
Transmembrane
Conductance Regulator
Small molecule drug…
…developed by Vertex…
…with support from the
Cystic Fibrosis Foundation…
…that works for CF patients
who have the G551D mutation.
http://learn.genetics.utah.edu/content/disorders/whataregd/cf/
CF is the most common
lethal genetic disease in Caucasians.
http://learn.genetics.utah.edu/content/disorders/whataregd/cf/
The CFTR Gene was first cloned and sequenced in 1989….
…… by Lap-Chee Tsui and Francis Collins
Riordan, J.R. et.al., Identification of the Cystic Fibrosis Gene: Cloning and
Characterization of Complementary DNA. Science, 245, pp1066-1073 (1989)
CFTR is a member of ATP-binding cassette (ABC) transporter superfamily.
Outside of cell
M
E
M
B
R
A
N
E
Six
Transmembrane
Alpha Helices
Six
Transmembrane
Alpha Helices
Cytoplasm
Nucleotide
Binding Domain
Nucleotide
Binding Domain
The first six transmembrane helices (TM1, TM2, TM3, TM4, TM5, TM6) of CTFR
If each TM sequence
(21 aa) is an alpha helix
– and there are 3.6
amino acids per turn of
the helix….each TM
helix would be ~6 turns
long. Since each turn of
the helix is 5.4A long….6
turns is 32 A or 3.2 nm.
This is just long enough
to span the thickness of
a lipid bilayer
The CFTR protein consists of the following domains….
CFTR mRNA map
N
C
TM 1-6
NBD1
Regulatory
Domain
TM 7-12
NBD2
2 important mutations map to the NBD of CFTR
G551D
is here
The F508del
is here
OMG! …. the F508del mutation of CFTR
Understanding the F508del mutation of CFTR is a mind-blowing experience!
Hang on……
507
The nucleotide sequence of the
normal gene in this region is:
While you might assume that the
F508 mutation is UUU – in fact it is
CUU.
509
----CAUCUUUGGUG------ I
F
G ---
3 base
deletion
----CAUCUUUGGUG---507
The CUU deletion changes the sequence to
508
508
----CAUUGGUG------ I
G ---
To put this in words,…. The 3rd position of codon 508 becomes the 3rd position
of codon 507. And since both AUC and AUU both encode Isoleucine (I) – this
3base deletion results in a clean deletion of F508, with no other change in
amino9 acid sequence. Is this a great country or what?
Potential PKA (Protein Kinase A) target sites in the R-domain of CFTR:
The Regulatory Domain of CFTR contains
many “dibasic” target sites that are
phosphorylated by Protein Kinase A
(PKA) and Protein Kinase C (PKC).
Shown below --- a listing of the dibasic
sites – and flanking sequence – found in
this region of CFTR.
As you “look for patterns” (a
Crosscutting Concept in the NGSS), can
you see a larger consensus sequence
that defines this phosphorylation site?
Sanger Sequencing Gel – of the region surrounding the F508 codon
Normal
CF
G A T C
G A T C
Riordan, J.R. et.al., Identification of the Cystic Fibrosis Gene: Cloning and
Characterization of Complementary DNA. Science, 245, pp1066-1073 (1989)
CFTR Model,… 1989
Riordan, J.R. et.al., Identification of the Cystic Fibrosis Gene: Cloning and
Characterization of Complementary DNA. Science, 245, pp1066-1073 (1989)
CFTR Model,… 2000
Akabas, M.H., Cystic Fibrosis Transmembrane Conductance Regulator J.
Biol. Chem. 275, pp3729-3732 (2000)
CFTR Model,… 2000
Akabas, M.H., Cystic Fibrosis Transmembrane Conductance Regulator J.
Biol. Chem. 275, pp3729-3732 (2000)
CFTR Model,… 2006
Gasby, D. et.al., The ABC protein turned
chloride channel whose failure causes cystic
fibrosis. Nature 440, pp477-483 (2006)
CFTR Model,… 2010
Atwell et.al., Structures of a minimal human CFTR first nucleotide-binding
domain as a monomer, head-to-tail homodimer, and pathogenic mutant. Prot.
Eng. Design & Selection 23, pp375-384 (2010)