Peripheral Protein Quality Control as a Novel Drug Target for CFTR Stabilizer

Conformationally defective cystic fibrosis transmembrane conductance regulator (CFTR) including rescued ΔF508-CFTR is rapidly eliminated from the plasma membrane (PM) even in the presence of a CFTR corrector and potentiator, limiting the therapeutic effort of the combination therapy. CFTR elimination from the PM is determined by the conformation-dependent ubiquitination as a part of the peripheral quality control (PQC) mechanism. Recently, the molecular machineries responsible for the CFTR PQC mechanism which includes molecular chaperones and ubiquitination enzymes have been revealed. This review summarizes the molecular mechanism of the CFTR PQC and discusses the possibility that the peripheral ubiquitination mechanism becomes a novel drug target to develop the CFTR stabilizer as a novel class of CFTR modulator.


INTRODUCTION
Cystic fibrosis (CF) is one of the most lethal autosomal-recessive diseases caused by mutation in CFTR (Lopes-Pacheco, 2016). CFTR mutations are classified as I-VII according to their properties (I-protein synthesis defect, II-maturation defect, III-gating defect, IV-conductance defect, V-reduced quantity, VI-reduced PM stability, VII-no mRNA transcription). The most prevalent CF causing mutation, F508, was classically categorized as class II mutation. However, rescued F508 (r F508)-CFTR by corrector (e.g., VX-809/lumacaftor) or low temperature culture shows class III and VI phenotypes (Dalemans et al., 1991;Veit et al., 2016a). Although drug targets of the class II or III mutations are well studied, that of the class VI mutation are not because the mechanism of CFTR PM stability regulation are still veiled by numerous undefined molecules involved in CFTR PQC system. In this review, we summarize accumulated findings regarding the CFTR PQC from the molecular and environmental aspects and also discuss the potential of recently identified PQC machineries including endocytic adaptors and ubiquitination enzymes as targets for CFTR stabilizer which anchors the functional channel at the PM and reduces the degradation (Figure 1).
The CFTR is internalized by clathrin-mediated endocytosis (CME) and recycled back to the PM. Conformationally defective CFTR produced by genetic mutations (e.g., F508, T70) and/or environmental stresses (e.g., heat) selectively undergoes ubiquitination at the PM by PQC machineries. The ubiquitinated CFTR is rapidly internalized and delivered to lysosome for degradation (Sharma et al., 2004;Okiyoneda et al., 2010).
Internalized CFTR could be de-ubiquitinated at endosomes by deubiquitinase (DUB) and recycled back to the PM depending on the conformational states. The class VI mutations render the CFTR unstable at the PM. Additionally, the class I, II and some class III CFTR mutants also show PM instability (Lukacs et al., 1993;Haardt et al., 1999;Silvis et al., 2003;Wang et al., 2014;Veit et al., 2016a). N-glycosylation, especially the core-glycosylation, determines the CFTR PM stability likely by affecting the CFTR conformational stability (Glozman et al., 2009;Cholon et al., 2010). Protein translation kinetics is also a significant factor that modulates proper cotranslational folding. Knock down (KD) of ribosomal protein L12 (RPL12) increases F508-CFTR PM expression and stability (Veit et al., 2016b). RPL12 KD might affect protein translation kinetics associated with co-translational protein folding efficiency (Buhr et al., 2016) and thereby improve CFTR thermodynamic stability which also determines the CFTR PM stability (Okiyoneda et al., 2010;Rabeh et al., 2012). Thus, correcting the CFTR structural defects at the ER could improve the PM stability.

Infection and Inflammation
The CFTR loss of function induces airway surface liquid (ASL) dysregulation which impairs clearance of infected bacteria and/or fungi, and increases the concentration of other soluble signal mediators such as cytokines, chemokines and growth factors. Pseudomonas aeruginosa (PA) is one of the most common bacteria found in CF respiratory tissue and responsible for lung injury in CF (Koch, 2002;Bhagirath et al., 2016). PA destabilizes PM CFTR by inhibiting endocytic recycling (Swiatecka-Urban et al., 2006). PA secretes CFTR inhibitory factor (Cif) that stabilizes complex formation of ubiquitin (Ub) specific peptidase 10 (USP10) and GTPase activating protein (SH3 domain) binding protein 1 (G3BP1) and inhibits CFTR-USP10 interaction. Cif inhibits internalized CFTR sorting to recycling pathway by suppressing USP10 dependent CFTR de-ubiquitination at endosome, resulting in the lysosomal degradation of WT-CFTR (Bomberger et al., 2011). PA also activates transforming growth factor β1 (TGF-β1) signaling that is an important modifier of lung disease severity in CF (Harris et al., 2011). TGF-β1 inhibits functional PM expression of WT-CFTR and F508-CFTR by reducing mRNA level (Snodgrass et al., 2013;Sun et al., 2014) although its role in the PQC remains unknown.

Heavy Metals
More than 10 ppb of arsenic induces the WT-CFTR ubiquitination and lysosomal degradation via c-Cbl in CF bronchial epithelial (CFBE) cells (Bomberger et al., 2012). Importantly, the phenotype of arsenic toxicity overlaps with CF patient (Bomberger et al., 2012;Mazumdar et al., 2015). Cadmium (Cd) is a major component of cigarette smoke (CS), and its inhalation is associated with decreased pulmonary function and chronic obstructive pulmonary disease. Cd reduces CFTR PM level, but it remains unknown if it reduces the PM stability (Rennolds et al., 2010).

Cigarette Smoke
Cigarette smoke is a major risk factor of chronic obstructive pulmonary disease and interferes with CFTR functionality. Ten minutes of CS exposure transiently suppresses CFTR function, induces internalization and decreases ASL height in human bronchial epithelial (HBE) cells (Clunes et al., 2012). CS promotes CFTR internalization in BHK cells and results in increased insolubility of CFTR and colocalization with vimentin, a filament protein associated with aggresome Ca 2+ dependently. This observation suggesting that CS induces PM CFTR destabilization by stimulating internalization and aggregation in addition to suppressing CFTR functionality (Clunes et al., 2012;Rasmussen et al., 2014).

Endocytosis Adaptors and Tethering Factors
Endocytosis is the critical step of elimination of PM CFTR as a part of PQC and is regulated by several molecules. WT-CFTR is internalized slowly by CME while misfolded r F508-CFTR endocytosis is accelerated (Sharma et al., 2004;Swiatecka-Urban et al., 2005;Varga et al., 2008;Okiyoneda et al., 2010). KD of CME adaptor AP-2 µ2 subunit or disabled 2 (DAB2) stabilizes r F508-CFTR at the PM by inhibiting endocytosis (Fu et al., 2012(Fu et al., , 2015. CFTR has a postsynaptic density 95, disks large, zonula occludens-1 (PDZ) binding motif at C-terminus and binds with Na + /H + exchanger regulatory factor (NHERF1) PDZ domain. NHERF1 tethers CFTR with Ezrin and works as a scaffold protein that supports CFTR efficient channel activation and apical PM localization (Favia et al., 2010;Arora et al., 2014;Loureiro et al., 2015). NHERF1 also binds to misfolded F508-CFTR and increases the PM stability by inhibiting carboxy terminus of HSP70-interacting protein (CHIP) Ub ligase interaction (Loureiro et al., 2015). An exchange protein directly activated by cAMP1 (EPAC1) selective activating cAMP analog 007-AM promotes WT-CFTR and NHERF1 interaction and increases CFTR PM stability in CFBE cells by suppressing endocytosis (Lobo et al., 2016). EPAC1 activation can rescue F508-CFTR PM expression, and its effect is further improved with VX-809 combination (Lobo et al., 2016).
Filamin-A (FLN-A) is a membrane tethered actin adaptor protein and interacts with CFTR N-terminus region. S13F mutation of CFTR compromises FLN-A binding and consequently destabilizes the PM CFTR (Thelin et al., 2007). FLN-A binds with both WT and r F508-CFTR at similar level, however, its contribution to the CFTR PQC remains unclear.

Chaperones
Molecular chaperones selectively interact with and stabilize unfolded or partially folded protein to acquire a functionally active conformation. Nascent CFTR interacts with a panel of chaperones and co-chaperones including HSC70, HSP70, HSP90, and CNX at the ER (Yang et al., 1993;Pind et al., 1994;Loo et al., 1998;Meacham et al., 1999;Okiyoneda et al., 2004). Even at the post-ER compartments, conformationally defective CFTR such as unfolded r F508-CFTR is recognized by chaperone/cochaperone complex (Okiyoneda et al., 2010). HSC70/HSP90 complex selectively interacts with unfolded r F508-CFTR at the post-Golgi and this interaction is crucial for the unfolding dependent ubiquitination (Okiyoneda et al., 2010). KD of HSC70/HSP90 complex (HSP90, HSC70, HOP, AHA1, DNAJB2, DNAJA1, BAG1) increases the r F508-CFTR PM stability in HeLa cells (Okiyoneda et al., 2010). The HSC70/HSP90 complex is also essential for maintaining kinetic and thermodynamic stability of r F508-CFTR at the PM by reshaping the CFTR conformation during energetic destabilization (Bagdany et al., 2017). This chaperone activity also maintains r F508-CFTR channel function at the PM (Bagdany et al., 2017). Thus, modulating the chaperone activity would be a viable target for attenuating the ubiquitination and for stabilizing the CFTR function at the PM.

Ubiquitination Enzymes
Ubiquitination determines CFTR elimination not only at the ER, but also from the PM. Ubiquitination is mediated by a sequential action of E1, E2, and E3 enzymes and this modification could be removed by DUB. Specifically, E3 Ub ligase has been proposed to determine the substrate specificity. CHIP is the first identified E3 ligase responsible for the CFTR PQC (Okiyoneda et al., 2010). Consistent with the action at the ER (Meacham et al., 2001), CHIP selectively interacts with unfolded F508-CFTR at the post-Golgi through the HSC70/HSP90 chaperones. CHIP KD reduces the ubiquitination of unfolded F508-CFTR, resulting in the decelerated endocytosis and lysosomal delivery in HeLa cells (Okiyoneda et al., 2010). CHIP KD also stabilizes r F508-CFTR at the PM of polarized CFBE cells (Fu et al., 2015).
E3 ligase c-Cbl may play a role in the CFTR peripheral QC, but its contribution could be modest since its KD slightly increases r F508-CFTR PM stability in CFBE cells Fu et al., 2015). c-Cbl also binds with WT-CFTR and decreases the PM stability without affecting the ubiquitination, suggesting that c-Cbl could regulate constitutive PM turnover of folded CFTR by inducing endocytosis through its C-terminus adaptor function (Ye et al., 2010).
A number of DUBs regulate the CFTR turnover. USP10, a DUB localized at early endosomes, interacts with WT-CFTR and reduces the CFTR poly-ubiquination in CFBE cells. The USP10mediated deubiquitination enhances the endocytic recycling of WT-CFTR (Bomberger et al., 2009). The role of USP10 in the PM stability of conformationally defective CFTR such as r F508-CFTR remains unclear.
Recently, we have discovered RING finger and FYVE like domain containing E3 Ub protein ligase (RFFL) as a novel component of the CFTR PQC machineries by a comprehensive siRNA screen in CFBE cells (Okiyoneda et al., 2018). RFFL selectively recognizes unfolded r F508-CFTR through the disordered regions. RFFL promotes K63-linked polyubiquitination of the unfolded CFTR in post-Golgi, resulting in accelerated endocytosis and lysosomal degradation. Importantly, RFFL directly interacts with conformationally defective CFTR such as r F508-CFTR, but not with folded WT-CFTR at the PM and endosomes. Moreover, the RFFL-mediated ubiquitination is conformation dependent as it selectively ubiquitinates thermally unfolded NBD1. RFFL KD enhances the functional PM expression of r F508-CFTR in the presence of VX-809, and this effect is further improved by inhibiting the HSC70-dependent ubiquitination machinery. Thus, RFFL plays an important role in the chaperone-independent CFTR PQC mechanism in HBE cell models.

CFTR MODULATORS AFFECTING THE CFTR PM STABILITY Pharmacological Chaperones and Chemical Chaperones
Pharmacological chaperones affect the CFTR PM stability by direct stabilization. CFTR corrector VX-809 is the first food and drug administration (FDA) approved CFTR corrector in combination with VX-770/ivacaftor (known as Orkambi). VX-809 selectively improves the processing of misfolded CFTR by stabilizing NBD1-membrane spanning domain (MSD) interface but not other misfolded proteins such as human ether-à-go-go-related gene (hERG) mutants (Van Goor et al., 2011;Okiyoneda et al., 2013;Ren et al., 2013;Farinha et al., 2015). VX-809 repairs not only the CFTR folding defect at the ER but also the CFTR PM instability. VX-809 washout prolongs F508-CFTR functional sustainability (Van Goor et al., 2011), suggesting that improvement of the CFTR folding at the ER could increase the thermal stability and proper co-and/or post-translational modifications that renders CFTR more energetic robust conformations even at the PM. VX-809 also promotes F508-CFTR and NHERF1 interaction, that may increase the PM stability (Arora et al., 2014). C3 (CFcor-325/VRT-325) and C4 (Corr-4a) also extend r F508-CFTR PM stability in CFBE cells probably by directing binding (Wang et al., 2007;Varga et al., 2008) although their effect could be not specific to the conformationally defective CFTR (Van Goor et al., 2011). Chemical chaperones such as glycerol also increases the r F508-CFTR PM stability probably by non-specifically improving the conformational stability (Okiyoneda et al., 2013).

CFTR Potentiators
The first FDA approved CFTR potentiator VX-770 improves the gating defect of some CFTR mutants. However, chronic VX-770 treatment destabilizes the PM r F508-CFTR in CFBE and F508 homozygous CF patient HBE (CF-HBE) cells (Cholon et al., 2014;Veit et al., 2014). Importantly, chronic VX-770 treatment diminishes the VX-809 therapeutic efficacy by stimulating the elimination of PM r F508-CFTR (Cholon et al., 2014;Veit et al., 2014). In addition to VX-770, several CFTR potentiators including P1 (VRT-532) and P2 (PG-01) also decrease the r F508-CFTR PM stability (Veit et al., 2014). VX-770 and other potentiators could destabilize a variety of CFTR rare mutants referred to as CFTR2 mutants including E92K and L1077P at the PM . Thus, several CFTR potentiators may decrease the thermal stability of metastable mutant CFTR at the PM by inducing conformational change that positively affects for channel gating but negatively affects stability. High-throughput screening has identified several novel CFTR potentiators such as class A analog 4 (A04) and class P analog 12 (P12) that could not destabilize the PM r F508-CFTR (Phuan et al., 2015).

Proteostasis Regulating Drugs
Proteostasis regulating drugs that affect array of proteins regulating CFTR folding and QC also affect the CFTR PM stability. Histone deacetylase (HDAC) inhibitor suberoylanilide hydroxamic acid (SAHA) alters expression of a subset of CF-interacting gene products (e.g., chaperones and DAB2) and sustains PM expression of F508-CFTR in CFBE cells (Hutt et al., 2010). Tissue transglutaminase (TGM2) inhibitor cystamine also stabilizes F508-CFTR at the PM of airway epithelial cells by restoring BECN1 interactome which is sequestrated by CFTR dysfunction (Luciani et al., 2012;Villella et al., 2013). MLK3 pathway inhibitor oxozeaenol has been reported to be effective in correcting the F508-CFTR proteostasis defect in the primary HBE cells (Trzcinska-Daneluti et al., 2012). Oxozeaenol could stabilize F508-CFTR at the PM as MLK3 KD reduces mature F508-CFTR elimination by PQC (Hegde et al., 2015).

Cavosonstat and CAL Inhibitor
HSP70/HSP90 Organizing Protein is adaptor protein which coordinates HSP70 and HSP90 function in protein folding and regulates CFTR maturation and PM stability (Odunuga et al., 2004;Okiyoneda et al., 2010). HOP S-nitrosylation by S-nitrosoglutathione (GSNO) induces HOP degradation and increases F508-CFTR PM expression (Marozkina et al., 2010). Levels of S-nitrosothiols such as GSNO are low in CF airway (Grasemann et al., 1999) and S-nitrosothiol decreases the internalization rate of r F508-CFTR in HBE cells (Grasemann et al., 1999(Grasemann et al., , 2000Zaman et al., 2014). Cavosonstat (N91115) is an orally bioavailable inhibitor of GSNO reductase and restores GSNO levels (Donaldson et al., 2017). Cavosonstat is the first CFTR stabilizer in phase II trials, but it was not beneficial for improvement of lung function in combination with ivacaftor.
CFTR-associated ligand binds CFTR via a PDZ interaction domain and targets CFTR for lysosomal degradation (Cheng et al., 2004). CAL inhibition increases the PM stability of F508-CFTR (Cushing et al., 2010) and cell penetrating CAL inhibiting peptide is established (Qian et al., 2015). CAL inhibitor has been developed as a cell surface CFTR stabilizer in pre-clinical level while its therapeutic efficacy and conformational selectivity remain unclear.

Ub Ligase Inhibitors
RING finger protein 5 (RNF5/RMA1) is an ER associated E3 Ub ligase that regulates early stage CFTR proteostasis at the ER (Younger et al., 2006). A RNF5 inhibitor Inh-2 identified by homology modeling and virtual ligand screening causes significant rescue of F508-CFTR in immortalized and primary HBE cells from CF patients (Sondo et al., 2018). Intriguingly, Inh-2 modestly increases mature F508-CFTR half-life and this stabilization effect is further improved by VX-809. While the contribution of RNF5 in the CFTR peripheral QC remains unclear, RNF5 inhibitor may be useful to overcome the CFTR instability.
Currently, CHIP and RFFL are the only Ub ligases responsible for the CFTR peripheral QC (Okiyoneda et al., 2010(Okiyoneda et al., , 2018. Thus, inhibiting their activity could selectively reduce the ubiquitination and elimination of unfolded CFTR from the PM, improving the limited efficacy of CF combination therapy. CHIP binds and regulates a number of substrates via chaperones (Connell et al., 2001). Moreover, inhibiting the CHIP activity induces deleterious effect as the CHIP KO mice result in the abnormal phenotypes including ataxia and pre-mature death 1 . In contrast, RFFL could bind and regulate a limited number of substrates because of its nature of direct binding to the CFTR through the disordered regions (Okiyoneda et al., 2018). More importantly, inhibiting the RFFL activity seems to have no venomousness since the RFFL KO mice exhibit no abnormal phenotype (Ahmed et al., 2009). Therefore, counteracting RFFL activity may provide a preferable therapeutic approach as a CFTR stabilizer that is a class of drugs that extends the PM resident time of CFTR class VI mutants. Although future studies 1 http://www.informatics.jax.org/marker/phenotypes/MGI:1891731 are needed to validate the impact on F508-CFTR in CF-HBE cells, developing agents selectively inhibiting RFFL-mediated CFTR ubiquitination may help improve the efficacy of CF pharmacological therapy.

CONCLUSION AND PERSPECTIVE
Beside the progresses of CF pharmacological therapy, stabilizing the cell surface CFTR remains challenging and is necessary to improve the limited therapeutic efficacy. Recent studies have revealed some of the CFTR PQC mechanism eliminating the functional but conformationally defective CFTR from the PM. Understanding the CFTR PQC mechanism help the development of the CFTR stabilizer, a novel class of CFTR modulator necessary to establish the robust CF pharmacological therapy.

AUTHOR CONTRIBUTIONS
All authors listed have made a substantial, direct and intellectual contribution to the work, and approved it for publication.

FUNDING
This work was supported by JSPS KAKENHI grant numbers JP25893275, 15H05643, and 15H01192.