Overview: Cystic Fibrosis

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Transcript Overview: Cystic Fibrosis

Treating Cystic Fibrosis Using SNitrosothiol Compounds
This study was designed to test the hypothesis that physiological concentrations of
naturally occurring S-Nitrosothiol compounds increase the expression and maturation
of the cystic fibrosis transmembrane conductance regulator (CFTR) protein in cystic
fibrosis and non-cystic fibrosis cells.
Cystic fibrosis (CF) is a genetic disorder caused by a mutation in the protein called
CF Transmembrane Conductance Regulator (CFTR), which regulates chloride
transport in epithelial cells. The most common mutation, ΔF508 CFTR results in a
deletion of the amino acid phenylalanine (F), at position 508, which causes the
protein to fold incorrectly. Wrongly folded CFTR proteins cannot pass through the
endoplasmic reticulum, are degraded by enzymes, and are unable to regulate
chloride transport in the cell membrane. This leads to poor salt regulation in
epithelial cells, which causes germ accumulation in the lungs and disrupts the
pancreas. S-Nitrosothiol compounds which naturally occur in the body are
hypothesized to aid CFTR maturation in cells. CF patients have lower levels of
these compounds due to having more enzymes called S-nitrosoglutathione
reductase. This experiment tested whether S-Nitrosothiols increase CFTR
maturation in human epithelial cells by using Western Blot analysis and
immunohistochemistry techniques. The results showed that the cells treated with SNitrosothiols had more expression and maturation of the CFTR protein than those
without S-Nitrosothiols. When the cell membranes were examined, it was seen that
the cells treated with S-nitrosoglutathione diethyl ester (GNODE), one class of S–
Nitrosothiols, had more maturation of the CFTR protein than those treated with Snitrosoglutathione (GSNO), another S–Nitrosothiol, which is likely due to the greater
membrane permeability of GNODE. Further testing will focus on the discovery of the
optimal dose of S-Nitrosothiols that can be given to patients with cystic fibrosis to
treat them.
17th Century German Saying
“” “Woe to that child which when kissed on the forehead tastes salty.
He is bewitched and soon must die”
 Cystic fibrosis affects more than 30,000 kids and young adults in the United States
and 70,000 worldwide. Kids who have it are more vulnerable to repeated lung
infections, which disturb the normal function of epithelial cells. When the CFTR
protein is defective, epithelial cells can not regulate the way that chloride ions pass
across cell membranes. This disrupts the essential balance of salt and water.
Because of that, mucus in the lung airways becomes very thick, sticky, and hard to
move. Also, cilia can not move properly and germs start to collect on the cells,
which leads to life-threatening lung infections. The mucus also blocks the
pancreas and stops enzymes from assisting the body with breaking down and
absorbing food (Figure 1).
A look inside cystic fibrosis
Figure 1.
From Jay Smith
Discover Magazine
 It has recently been discovered that there are some small naturally occurring
compounds that are normally present in the human airway. These compounds are
capable of increasing the maturation of the commonly identified mutated form of
the cystic fibrosis transmembrane conductance regulatory (ΔF508 CFTR) protein.
These are known as S-Nitrosothiols and are formed in the human airways from a
reaction between nitric oxide and intracellular glutathione. These compounds
appear to cause chemical reactions that allow CFTR to mature and become active
on the cystic fibrosis cell surface. Remarkably, researchers have found that these
compounds are reduced in the CF airways.
 Researchers have previously shown that replacement therapy with low doses of
inhaled S-Nitrosothiols are well-tolerated and improve oxygen levels in patients
with cystic fibrosis. However, evidence also suggests that excess concentrations of
these compounds in the CF airway could have unwanted effects, perhaps even
inhibiting CFTR maturation and function. Further, the mechanisms by which SNitrosothiols exert beneficial effects are not clear. Reseachers hope to identify both
the ideal level of S-niyrosothiols and lowest effective dose, suitable for clinical trials
for cystic fibrosis.
Overview: Cystic Fibrosis
mutant mother
normal phenotype
mutant father
normal phenotype
CF phenotype
Figure 2.
normal phenotype
normal phenotype
normal phenotype
Overview: Cystic Fibrosis
 Human CFTR gene was first identified in 1989 and is located on the long
arm of chromosome 7
 There are almost 2000 mutations but the most common mutation is
known as ΔF508 CFTR
 This mutation occurs due to the deletion of the three nucleotides which
produce the amino acid phenylalanine at position 508
 Figure 3 shows a model of the CFTR protein structure. The CFTR protein is
made up of 5 domains
 Two nucleotide binding domains (NBD1/NBD2), that bind and
hydrolyze ATP to get energy
 Two dual sets of membrane-spanning domains (MSD1/MSD2) that
form the chloride ion channels
 A central regulatory domain (R) that regulates the CFTR function
 In normal cells, the CFTR protein allows the release of chloride ions from
the cell. If CFTR doesn’t function, chloride ions cannot leave the cell
 Without the balance of chloride ions, water does not exit the cell and
without water, mucus thickens outside the cell
 Cilia also cannot beat properly and bacteria starts to collect on the cells
which leads to infection
 Presently, there is no cure for CF and current treatment only keeps it
under control
Model of CFTR Protein Structure
Figure 3.
Sawczak, V. et al. 2015
Current Drug Targets 16: 1-15
S-Nitrosothiols Synthesis
Figure 4. Nitric oxide (NO) is synthesized by the oxidation of amino acid, L-arginine
to the nitric oxide and L-citruline. The reaction is catalyzed by enzymes called nitric
oxide synthase (NOS). Nitric oxide in turn, is involved in the production of SNitrosothiol compounds, including S-Nitrosoglutathione. S-nitrosoglutathione
reductase (GSNOR) is found in high levels in CF cells and it catalyzes the
degradation of GSNO in the cells.
Overview: S-Nitrosothiol Compounds
S- nitrosoglutathione (GSNO)
S-nitroso-N-acetyl cysteine (SNOAC)
S- nitrosocysteinyl glycine (CGSNO) S- nitrosoglutathione diethyl ester (GNODE)
Figure 5.
Zaman, K. et al. 2013
Current Pharmaceutical Design 19: 3509-3520
Cell lines
 Normal Human Bronchial Airway Epithelial cells
 Mutant ΔF508 CFTR Human Bronchial Airway Epithelial cells
(provided by Dr. Eric Sorscher from the University of Alabama)
 HBAE cells were grown in DMEM (Dulbecco’s Modified Eagle Medium).
 Medium contained 10% FCS (fetal calf serum) and 1% penicillin/streptomycin.
 Cells were maintained at 370C in a humidified atmosphere of 5% CO2 (carbon
dioxide) and air.
Western blot analysis.
1. Whole cell extracts were prepared in 1% NP-40 lysis buffer (50 mM Tris-HCl. pH
8.0, 1% NP-40, 150 mM NaCl, 2 µM leupeptin, 1 µM aprotinin, and 1 µM pepstatin,
1mM DTT, 1 µM PMFS, and 2 µM Na3VO4).
2. Insoluble material from NP-40 was recovered and sheared by passage through a
25-gauge needle.
3. Protein was quantitated by the Lowry assay by using a protein assay kit (Sigma
Chemical Co., St. Louis, MO).
4. One hundred micrograms of protein were fractionated on a 6% SDS
polyacrylamide gel in 1X Electrode Buffer (25 mM Tris, 192 mM glycine, 0.1% SDS
at pH 8.3).
5. The fractionated proteins were transferred to nitrocellulose membranes (Bio-Rad,
Hercules, CA) using an electrophoretic transfer cell with Tobin Transfer Buffer (25
mM Tris, 192 mM glycine, 20% methanol at pH 8.3).
6. Blots were blocked in Tris buffered saline-Tween 20 (TBS-T) (TBS-T = 10 mM TrisHCl, pH 8.0, 150 mM NaCl, 0.05% Tween 20) containing 5% nonfat dried milk.
7. Blots were probed with a 1:1000 dilution in TBS-T containing 5% nonfat dried milk
for 45 min at room temperature.
8. Blots were washed several times in TBS-T, incubated for 30 min with a 1:2000
dilution of (HRP)-conjugated anti-mouse antibody (Bio-Rad, Hercules, CA) in
TBS-T containing 5% nonfat dried milk for 30 min.
9. Blots were washed as previously described and CFTR proteins were visualized
by enhanced chemiluminescence (ECL, Amersham) using Hyperfilm
(Amersham Pharmacia Biotech).
Cell surface biotinylation.
1. HBAE cells were treated for 4 h without or with different concentrations of
2. The cells were washed (x3) with ice-cold phosphate buffered saline (pH 7.4)
containing 0.1 mM CaCl2 and 1 mM MgCl2 (PBSCM) and then treated in the
dark with PBSCM buffer containing 10 mM sodium periodate for 30 min at 20oC
3. The cells were washed (x3) with PBSCM and biotinylated by treating with
sodium acetate buffer (100 mM sodium acetate buffer, pH 5.5; 0.1 mM CaCl2
and 1 mM MgCl2) containing 2 mM biotin-LC hydrazide (Pierce, Rockford, IL) for
30 min at 20oC in the dark. The cells were then washed (x3) with sodium
acetate buffer and solubilized with lysis buffer containing Triton X 100 and
protease inhibitors.
4. CFTR was Iimmunoprecipitated and subjected to SDS-PAGE on 6% gels;
biotinylated CFTR was detected with streptavidin-conjugated horseradish
S-Nitrosothiols increased maturation of CFTR
Band B
Band C
Band C
Band B
Band C
GSNO (µM) 0
Band C
Figure 5. S-Nitrosothiol compounds increase the expression of defective
ΔF508 CFTR expression and maturation in primary human airway
epithelial cells (A and B) and in the cell surface (C and D).
Treatment with SNO increased defective
CFTR expression on the cell surface
GSNO (10 µM)
Figure 6. Immunohistochemistry of human bronchial airway epithelial
cells with the anti-CFTR (mAb 596) antibody. Cells were treated with 10
µM GSNO (A) and a control (no GSNO) for 4 hours.
µM NADH/min/mg
GSNO reductase activity differences in normal and
defective ΔF508 CFTR cells
Norma ΔF508 CFTR
S-Nitrosoglutathione Reductase (GSNO-R)
activity is significantly elevated in the HBAE cells
Expressing defective ∆F508 CFTR whereas compared to the normal HBAE cells
Figure 7. S-Nitrosoglutathione Reductase (GSNO-R) immunostaining is significantly elevated in the HBAE
cells expressing defective ∆F508 CFTR whereas compared to the normal HBAE cells
 Here, it was found that both forms of immature and mature defective ΔF508
CFTR protein were markedly induced by SNO compounds in human
bronchial airway epithelial cells
 S-Nitrosothiols increased defective ΔF508 CFTR expression on the cell
membrane of primary human bronchial airway epithelial cells
 In addition, it was shown that GSNO reductase activity and expression is
significantly elevated in the defective ΔF508 CFTR human bronchial airway
epithelial cells when compared to the normal human bronchial airway
epithelial cells
Proposed model of interactions between
GSNO, chaperones and CFTR
Transport to Plasma
Golgi Apparatus
CFTR Glycosylation
Endoplasmic Reticulum
Protein Folding
mRNA Translation
Proteosomal Degradation
mRNA Transcription
CFTR gene
Figure 8.
Sawczak, V. et al. 2015
Current Drug Targets 16: 1-15
The present data suggests that S-Nitrosothiols at physiological concentrations
increase CF and non-CF CFTR expression and maturation
It is speculated that these novel observations will be critical to optimizing the
dosing of SNOs that might be used to improve management of CF patients
Zaman, K., McPherson, M., Vaughan, J., Hunt, J.F., Mendes, F., Gaston, B. and Palmer, L.A. (2001). S-Nitrosoglutathione increases cystic fibrosis
transmembrane regulator maturation. Biochem.Biophys.Res.Commun. 284: 65-70.
Snyder, A., McPherson, M., Hunt, J.F., Johnson, M., Stamler, J.S. and Gaston, B. (2002). Acute effects of areosolized S-Nitrosoglutathione in cystic
fibrosis. Am.J.Respir.Crit.Care Med. 165: 1-5.
Howard, M., Fischer. H., Roux, J., Santos, B., Gullans, S., Yancey, P. and Welch, W. (2003). Mammalian osmolytes and S-Nitrosoglutathione
promote  F508 CFTR maturation and function. J.Biol.Chem. 278: 35159-35167.
Chen, L., Patel, R.P., Teng, X., Bosworth, C.A., Lancaster, J.R. and Matalon, S. (2006). Mechanisms of cystic fibrosis transmembrane conductance
regulator activation by S-Nitrosoglutathione. J.Biol.Chem. 281: 9190-9199.
Zaman, K., Carraro, S., Doherty, J., Henderson, E., Lendermon, E., Liu, L., Verghese, G., Zigler, M., Ross, M., Park, E., Palmer, L., Doctor, A.,
Stamler, J. and Gaston, B. (2006). S-Nitosylating agents: A novel class of compounds that increase cystic fibrosis transmembrane conductance
regulator expression and maturation in epithelial cells. Mol.Pharmacol. 70: 1435-1442.
Marozkina, N., Yemen, S., Borowitz, M., Liu, L., Plapp, M., Sun, F., Islam, R., Erdmann-Gilmore, P., Townsend, R., Lichti, C., Mantri, S., Clapp, P.,
Randell, S., Gaston, B. and Zaman, K. (2010). Hsp70/Hsp90 organizing protein as a nitrosylation target in cystic fibrosis therapy. Proc Natl Acad
Sci USA 107: 11393-11398.
Sawczak, V., Gesty, P., Zaidi, A., Sun, F., Zaman, K. (2015). Novel approaches for potential therapy of cystic fibrosis. Current Drug Targets 16: 115.