Mechanisms of CFTR activation by S-nitrosoglutathione (normal and CF)

Mechanisms of CFTR activation by S-nitrosoglutathione (normal and CF)

Cystic fibrosis (CF) is a multisystem disease associated with mutations in the gene encoding the CF transmembrane conductance regulatory (CFTR) protein [1]. 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 [2], [3]. The majority of wild-type (wt) CFTR, and virtually all deltaF508 CFTR, is degraded before reaching the cell surface [4], [5]. Certain agents and conditions increase expression, maturation, and function of deltaF508 CFTR [6].

S-Nitrosoglutathione is an endogenous bronchodilator and signaling molecule [7] that enhances expression, maturation, and function of both wt and deltaF508 CFTR in epithelial cells [8], [9], [10]. S-Nitrosoglutathione is present endogenously on the apical side of airway epithelium. It increases ciliary beat frequency, thereby improving mucociliary clearance [11].

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 [12], [13], [14].

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 [13].

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 [13], [15], [16], [17], [18].

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 [19]. 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) [20].

Ceruloplasmin also may catalyze the synthesis of S-Nitrosoglutathione [14], [21], [22].

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)) [11], [22], [23], [24], [25], [26], [27], [28].

Gamma GT can be involved in CFTR activation. S-Nitrosocysteinylglycine, the product of S-Nitrosoglutathione cleavage by Gamma GT, can increase DeltaF508 CFTR maturation [29].

SOD1, TXNRD1, TXNRD2 and TXNRD3 catabolize S-Nitrosoglutathione to form free NO radicals [11], [22], [23], [28]. Free NO can spontaneously react with Superoxide anion radical (O(2)(-)) to produce Peroxynitrite (ONOO(-)) [30]. The presence of SOD1, that catalyzes the dismutation of O(2)(-), can outcompete the peroxynitrite reaction. Cells may contain sufficient SOD1 to prevent inactivation of NO by O(2)(-) [12].

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 [29].

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) [9] and partly post-translational [8], [29]. For SP1, the additional mechanism for enhanced DNA-binding involves cysteine S-nitrosylation in the SP1 zinc finger-binding domain [9].

On the other hand, S-Nitrosoglutathione at nitrosative stress levels (100 microM) inhibits SP3 binding, augments competitive binding of SP1 and inhibits CFTR transcription [9].

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 [8], [29].

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 [31], [32], [33].

HSP90 (HSP90 alpha and HSP90 beta) and Heat shock 70kDa protein 8 (HSC70) are S-nitrosylated by S-Nitrosoglutathione, followed by CFTR folding and stabilization [29].

S-Nitrosoglutathione also 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 Csp. S-Nitrosoglutathione actually increases Csp expression (primarily post-transcriptionally) leading to increase in CFTR folding and maturation [29].

In the absence of S-Nitrosoglutathione, Csp initiates activation of HSC70 ATPase activity, which leads to CFTR degradation [34], [35], [36]., 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 [29].

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