Mechanisms of CFTR activation by S-nitrosoglutathione (normal
Cystic fibrosis (CF) is a multisystem disease associated with mutations in the gene
encoding the CF transmembrane conductance regulatory (CFTR)
protein . CFTR has several functions but is
typically regarded as an apical membrane Cl- channel in epithelial cells. Its
post-translational processing involves complex and incompletely defined series of
interactions with variety of chaperones and co-chaperones that assist in proper folding,
of CFTR, as well as its glycosylation, and assess the folded
protein it for possible defects. The most common mutation associated with CF, deltaF508,
results in a single amino acid deletion , . The majority
of wild-type (wt) CFTR, and virtually all deltaF508
CFTR, is degraded before reaching the cell surface , . Certain agents and conditions increase expression,
maturation, and function of deltaF508 CFTR .
S-Nitrosoglutathione is an
endogenous bronchodilator and signaling molecule  that enhances
expression, maturation, and function of both wt and deltaF508
CFTR in epithelial cells , , . S-Nitrosoglutathione is present
endogenously on the apical side of airway epithelium. It increases ciliary beat
frequency, thereby improving mucociliary clearance .
Nitric oxide synthases (NOSs) are involved in conversion of
L-arginine into nitric oxide (NO).
NO, in turn, is involved in production of S-nitrosothiols,
including S-Nitrosoglutathione. NO
may react directly with thiyl radicals or with thiols to form
S-nitrosothiols or S-nitrosothiol radicals, respectively , , .
In human, there are three isoforms of NOS: neuronal NOS
(nNOS), endothelial NOS (eNOS)
and inducible NOS (iNOS). nNOS
and eNOS are constitutively expressed and produce small
amounts of NO, whereas iNOS
expression is mainly induced by inflammatory stimuli. Induced iNOS synthesizes relatively
large quantities of NO. All three isoforms are expressed in
human airways .
In the case of CF, airway epithelial cells are more susceptible to bacterial and viral
infection due to impairment of the host NO defense pathway. Polymorphisms of constitutive
NOS (nNOS and
eNOS) and reduced iNOS
expression contributes to decreased NO production along with
bacterial consumption , , , , .
S-nitrosylation can functionally regulate the general activities of Heat shock protein
90kDa alpha (HSP90 alpha) and provide a feedback mechanism
for limiting eNOS activation.
S-Nitrosoglutathione covalently modifies a susceptible
cysteine residue in the HSP90 alpha domain that interacts
with eNOS. On the one hand,
S-nitrosylation abolishes the positive regulation on eNOS
activity mediated by native chaperone HSP90 alpha . On the other hand, direct S-nitrosylation can increase the activity of each of
the major forms of nitric oxide synthases (nNOS,
eNOS and iNOS) .
Ceruloplasmin also may catalyze the synthesis of
S-Nitrosoglutathione , , .
S-Nitrosoglutathione can be
catabolized by a number of enzymes, including Cu/Zn superoxide dismutase
(SOD1), gamma glutamyl transpeptidase (Gamma
GT), thioredoxin reductases (TXNRD1,
TXNRD2 and TXNRD3) and
glutathione-dependent formaldehyde dehydrogenase (ADHX
(GSNOR)) , , , , , , , .
Gamma GT can be involved in
S-Nitrosocysteinylglycine, the product of
S-Nitrosoglutathione cleavage by Gamma
GT, can increase DeltaF508 CFTR maturation
TXNRD2 and TXNRD3
catabolize S-Nitrosoglutathione to form free
NO radicals , , , . Free NO can spontaneously react
with Superoxide anion radical (O(2)(-)) to produce
Peroxynitrite (ONOO(-)) . The presence of
catalyzes the dismutation of O(2)(-), can
the peroxynitrite reaction. Cells may contain sufficient SOD1
to prevent inactivation of NO by
S-Nitrosoglutathione at low
micromolar concentrations increases the DeltaF508 and wild-type
CFTR expression and maturation.
S-Nitrosoglutathione mainly acts independently of the
classic NO radical/cyclic GMP pathway .
The effect of S-Nitrosoglutathione at 1-10 microM
concentration is partly transcriptional (it acts via increasing transcription factors
SP1 and SP3 expression and
their DNA-binding capacity)  and partly post-translational , . For SP1, the additional
mechanism for enhanced DNA-binding involves cysteine S-nitrosylation in the
SP1 zinc finger-binding domain .
On the other hand, S-Nitrosoglutathione at nitrosative
stress levels (100 microM) inhibits SP3 binding, augments
competitive binding of SP1 and inhibits
CFTR transcription .
Post-translational effect of S-Nitrosoglutathione is
associated with both increased expression and covalent modification - namely
S-nitrosylation - of proteins involved in CFTR folding, and
stabilization resulted in an increased CFTR maturation
ER-associated pathways of CFTR folding are affected by
chaperones and co-chaperones such as cytosolic Heat shock proteins 70 and 90kDa
(HSP70 and HSP90), DnaJ homolog
subfamily B member (Hdj-1) and others , , .
HSP90 (HSP90 alpha
and HSP90 beta) and Heat shock 70kDa protein 8
(HSC70) are S-nitrosylated by
by CFTR folding and stabilization .
increases expression of DnaJ homolog, subfamily C, member 5
(Csp) to enhance the association between
Csp and CFTR in the ER and
Golgi. S-Nitrosoglutathione does not S-nitrosylate
actually increases Csp expression (primarily
post-transcriptionally) leading to increase in CFTR folding
and maturation .
In the absence of S-Nitrosoglutathione,
Csp initiates activation of
HSC70 ATPase activity, which leads to
CFTR degradation , , ., This degradation is inhibited in the presence of
S-Nitrosoglutathione, allowing increased
Csp to continue stabilization of
CFTR. HSC70 has a single
critical cysteine residue in its ATP binding domain. S-nitrosylation of this cysteine
allows Csp to augment CFTR
folding without leading to CFTR degradation .
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