Putting CHIP(s) on the Table: Introducing Nitrosothiols into the Arena of CFTR Modulation.

Cystic fibrosis (CF) is an autosomal-recessive, genetically based, life-limiting disease characterized by viscid secretions in multiple organ systems (1, 2). The cystic fibrosis transmembrane conductance regulator gene (CFTR), on chromosome 7, codes for a cAMP-regulated chloride channel on multiple epithelial surfaces, including the airways, pancreas, and intestine (1, 2). Mutations can affect the CFTR protein at any point, including during formation, maturation, intracellular trafficking, surface function, and denaturation (1, 2). Over 2,000 mutations have been described, with a phenylalanine deletion (genetically: c.1521_1523delCTT) resulting in misfolded protein and accelerated degradation (F508del) being the most common disease-causing mutation (1, 2). Defective CFTR function impairs chloride conductance, with associated defective sodium transport across epithelial surfaces resulting in the generation of viscid secretions that are the hallmark of CF (1).

Multidisciplinary center-based care combined with therapeutic advances has dramatically increased life expectancy for people with CF (PWCF); indeed, infants born today are predicted to survive for almost 50 years (2). Precision therapies capable of modulating the underlying cellular defect (CFTR modulators) represent the most significant therapeutic advance in CF (2). With early diagnosis through newborn screening, and the development of CFTR modulators, clinical care is moving away from managing downstream complications and toward targeted therapies to prevent disease progression (1–3).

The first CFTR modulator, ivacaftor, is a CFTR potentiator that improves channel gating at epithelial surfaces and potentiates chloride conductance and CFTR function, with resultant clinical improvement (4). Only 4% of PWCF (2) who carry “gating” mutations are responsive to ivacaftor; in these individuals a sufficient quantity of the defective CFTR traffics to the cell membrane (2, 4) to provide substrate for ivacaftor (1, 2, 4). Combination therapy, which involves using a CFTR corrector such as lumacaftor or tezacaftor to promote CFTR production, with ivacaftor to potentiate channel gating, has widened the range of mutations amenable to CFTR modulation, including F508 del (3). The clinical benefits of current combination therapy require optimization (2, 5). Although early results of triple combination therapy (two correctors and one potentiator) appear promising, the search for CFTR modulators suitable for all PWCF continues (2, 3, 5). Gene agnostic CFTR amplifiers, which increase the protein substrate for CFTR modulators, may widen the scope for future combination therapies (5). Even with a portfolio of promising preclinical and clinical studies, better insight into molecular pathways associated with CFTR trafficking may provide necessary additional therapeutic targets (2, 5).

In this issue of the Journal, Zaman and colleagues (pp. 765–775) describe a novel alternative to current approaches for modulating CFTR (6). Whereas correctors, potentiators, and amplifiers all target the promotion of CFTR production (2, 5), Zaman and colleagues focus on reducing ubiquitination and proteasomal degradation, increasing CFTR trafficked to the cell membrane (6). The F508del CFTR mutation impairs protein folding and assembly, and is detected by the cells’ quality control (QC) system. Polyubiquitination of defective CFTR leads to its extraction from the endoplasmic reticulum (7). Detection of defective CFTR in the cytoplasm is performed by cHSP-70 (8). cHSP-70 interacts with the E3 ligase carboxy terminus of heat shock protein 70-interacting protein (CHIP), which in association with the E2 ligase UbcH5a effects ubiquitination and transport of misfolded CFTR for degradation by the cells’ QC system (the ubiquitin-proteasome system) (7, 8). Targeted therapies could inhibit ubiquitin-dependent degradation of CFTR, shielding it from the ubiquitin-proteasome system machinery, and CHIP inhibition has been identified as a potential pharmacological target (9) (Figure 1).

Figure 1.

Modulation of the cystic fibrosis transmembrane conductance regulator (CFTR): through the action of the CHIP (carboxy terminus of heat shock protein 70–interacting protein), misfolded CFTR (F508del) is targeted toward proteasomal degradation (left). CFTR correctors enhance CFTR transcriptional and translational output, allowing for some CFTR to be trafficked to the apical surface. Here, CFTR potentiators enhance the channel gating, resulting in chloride conductance (right). In the future, CFTR amplifiers could increase the CFTR substrate available for CFTR correctors. Alternatively, nitrosylation of CHIP (nitrosothiols) leads to reduced CHIP expression and activity, allowing misfolded CFTR to escape the quality control system, enhancing CFTR maturation. The addition of CFTR potentiators may provide further benefits. CHIP-NO = nitrosylated CHIP; cHSP-70 = cytosolic heat protein-70; Cl− = chloride anion; SNO = S-nitrosothiols; UPS = ubiquitin-proteasome system; Sp1/Sp3 = transcription factors specificity protein 1/specificity protein 3.

Nitric oxide (NO) synthases are abundant in the respiratory epithelium and are essential for the formation of stable bioactive S-nitrosothiols (SNOs), which are important for cellular signaling, -NO transfer, and regulation of protein function (10). Dysregulation of SNO formation is implicated in multiple respiratory diseases (10), including CF. Nitrosylation inhibits E3 ligases (11) and increases CFTR maturation (12) and chloride transport (13) in CF airway epithelial cells. Zaman and colleagues hypothesized that SNOs could inhibit CFTR proteasomal degradation through S-nitrosylation–dependent inhibition of CHIP. They showed CHIP expression in primary cells and bronchial cell lines, with dose-dependent S-nitrosylation of CHIP after treatment with S-nitrosoglutathione (GSNO), which reduced CHIP expression and activity. This led to a reduction in CHIP-CFTR and to decreases in CFTR ubiquitination, allowing F508 del CFTR to escape the endoplasmic reticulum QC system and traffic to the cell membrane. This reveals, for the first time, the mechanism behind the effect of S-nitrosylation on CFTR expression (Figure 1). Of all SNOs tested, S-nitrosoglutathione diethyl ester (GNODE) showed the highest effect on CFTR maturation, but in the current study this was tested only in cell monolayers. To progress toward CFTR-modulating therapies, cell-permeable GSNO analogs, including GNODE, should be used to test whether these findings can be replicated in differentiated pseudostratified epithelium. The bioavailability and bioactivation of SNOs may be lower in pseudostratified epithelium, potentially reducing the overall effect of GSNO on CFTR expression (14).

Modulation of the QC system may increase CFTR trafficked to the cell surface (6, 7), but the impact on CFTR-dependent chloride transport and later denaturation requires further investigation both in vitro and in vivo (2, 5). Previous in vivo studies using SNOs in CF focused on their antiinflammatory, smooth-muscle relaxant and bronchodilatory potential (15). A randomized trial of inhalational GSNO demonstrated tolerability, but only modest effects on oxygen saturation were seen (15). Although Zaman and colleagues focus on the potential of GSNO as an inhalational agent, all current CFTR modulators are delivered orally to permit systemic CFTR modulation (3, 4). CF is a multisystem disease and the systemic effects of CFTR modulators contribute significantly to their clinical benefits (1–5). Future studies are needed to investigate pharmacological modifications of QC systems and their effects on proteasomal degradation of CFTR, which may differ depending on cell- and tissue-dependent expression (7). With nitrosylation of CHIP being dose dependent (6), optimizing therapeutic ranges across multiple cell types and individuals will be challenging. Furthermore, any adverse effects of nitrosative stress (excessive NO/reactive oxygen species) that can contribute to NO-induced neurotoxicity (11) must be identified.

This study highlights a novel therapeutic target, nitrosylation of CHIP, with the potential to provide an innovative means of modulating CFTR. Selective modification of the QC machinery remains an attractive approach. Further exploration of therapeutic targets in the pathway identified by Zaman and colleagues may lead to the generation of systemic therapies that benefit PWCF. In combination with other CFTR modulators, this could further optimize clinical benefits in the future (Figure 1). Zaman and colleagues may have brought a potential new player to the table, and further study will determine what impact this has on those already there.