- Email: [email protected]
Protein and lipid interactions – Modulating CFTR trafficking and rescue
JCF-01544; No of Pages 5
Journal of Cystic Fibrosis xx (2017) xxx – xxx www.elsevier.com/locate/jcf
Original Article
Protein and lipid interactions – Modulating CFTR trafficking and rescue Carlos M. Farinha a,⁎, Elizabeth Miller b , Nael McCarty c a
University of Lisboa, Faculty of Sciences, BioISI - Biosystems & Integrative Sciences Institute, Campo Grande, 1749-016 Lisboa, Portugal b MRC Laboratory of Molecular Biology, Cambridge, UK c Emory University School of Medicine and Children's Healthcare of Atlanta, Atlanta, GA, USA Received 8 July 2017; accepted 28 August 2017
Abstract Different levels of CFTR regulation in the cell contribute to a stringent control of chloride secretion in epithelia. Tuning of chloride transport is achieved by modulating CFTR biogenesis, exit from the endoplasmic reticulum, trafficking, membrane stability and channel activity. In this short review, we summarize recent findings identifying interactions with other proteins – directly or through membrane lipids – and briefly discuss how these observations can provide clues to the design of better therapeutic approaches. © 2017 Published by Elsevier B.V. on behalf of European Cystic Fibrosis Society. Keywords: Endoplasmic reticulum; Membrane stability; cAMP; Lipids; Sphingomyelin
1. Introduction Understanding CFTR folding, trafficking and function has been a matter of great priority in the search for improved therapeutic approaches for patients with CF. After co-translational insertion in the membrane of the endoplasmic reticulum (ER) and core-glycosylation, CFTR undergoes a complex succession of steps with the main goal of checking the overall quality of its conformation that will ultimately lead to ER exit and traffic through the secretory pathway [1–3]. Failure to pass the various checkpoints of ER quality control targets the most frequent disease-causing mutant protein (F508del-CFTR) for premature degradation. If folding assessment at the ER allows CFTR to traffic, the protein passes through the Golgi to finally reach the plasma membrane where its stability and function as a protein kinase A (PKA) phosphorylation-regulated chloride channel are finely tuned. Interactions with other proteins but also with membrane lipids are crucial in regulating CFTR's life cycle. This
⁎ Corresponding author at: Departamento de Química e Bioquímica, Faculdade de Ciências da Universidade de Lisboa, Campo Grande, 1749-016 Lisboa, Portugal. E-mail address: [email protected] (C.M. Farinha).
short review will highlight some novel topics on CFTR regulation by different interactions.
2. Gene interactions regulating CFTR exit from the ER Targeting of F508del-CFTR to degradation takes place at the ER as a result of an intricate series of events that constitute a quality control surveillance system within the secretory pathway. Several studies have characterized different checkpoints assessing CFTR biogenesis in the ER [1,2,4,5], but the mechanistic basis for this surveillance is still not fully understood. Recent work has used a systematic approach to discover proteins that influence the biogenesis of misfolded ABC transporters more broadly using yeast as a model organism and Yor1 as a model protein [6]. Yor1 acts at the plasma membrane as a drug pump to confer resistance to oligomycin and, as in CFTR, misfolding mutations cause ER retention and proteasomal degradation, leading to oligomycin sensitivity [6]. The F508del-CFTR equivalent is F670del-Yor1 that results in misfolding and reduced half-life. To gain insight into the gene interaction network that regulates ER exit, double mutants for Yor1 and 5000 other genes were assessed based on alteration in the dose response to growth inhibition by oligomycin
http://dx.doi.org/10.1016/j.jcf.2017.08.014 1569-1993© 2017 Published by Elsevier B.V. on behalf of European Cystic Fibrosis Society. Please cite this article as: Farinha CM, et al, Protein and lipid interactions – Modulating CFTR trafficking and rescue, J Cyst Fibros (2017), http://dx.doi.org/10.1016/ j.jcf.2017.08.014
2
C.M. Farinha et al. / Journal of Cystic Fibrosis xx (2017) xxx–xxx
(normally extruded by Yor1, a process defective in the mutant strain). Such approaches evidenced that yeast gene interactions affecting the biogenesis of F670del-Yor1 were representative of human homologs previously found to modulate the processing of F508del-CFTR in mammalian cells – including syntaxins, Rab proteins, and ERQC proteins [6]. Interestingly, this study determined that the biogenesis of such mutants is positively influenced by the ER membrane complex (EMC) [7], an observation that was validated biochemically by knocking down an EMC ortholog in a human cell line expressing F508del-CFTR. The EMC complex seems to be involved in the detection of protein misfolding at the earliest stages of protein synthesis. Such detection generates feedback to the ribosome that halts or slows translation, serving to either promote folding or prevent aberrant proteins from entering the secretory pathway. These observations are in agreement with data identifying a role for the ribosomal stalk protein (RPL) 12 in CFTR biogenesis. Originally identified in a yeast screen also using F670del-Yor1, RPL12 was shown to influence CFTR elongation rate, with its knock-down enabling partial rescue of F508del-CFTR mislocalization and ER retention [8]. Phenomic analysis using yeast models has also contributed to a further validation of the ER exit sites as an essential QC checkpoint for CFTR. Incorporation of this channel (as well as other secretory proteins) into ER-derived vesicles involves recognition of cytosolic signals by the COPII coat protein, Sec24. In CFTR, this process was previously shown to involve the recognition of a diacidic exit code DAD (at residues 565–567), whose abrogation impairs CFTR trafficking, without a major effect on its folding [3,9]. In addition to export motifs in cargo proteins, ER export receptors are also involved in regulating cargo diversity and recruitment into vesicles. Among these, Erv14 (ER-derived vesicle protein of 14 kDa) mediates export of transmembrane proteins despite their potential to directly interact with Sec24. Using a targeted mutagenesis approach, this protein was recently shown to interact with client proteins through conserved residues in its second transmembrane domain [10]. This interaction allows Erv14 to function as a canonical cargo receptor coupling membrane proteins, including the CFTR homolog Yor1, to the COPII component Sec24. Interestingly
however, such interaction seems to work on a dual mode, involving cargo and coat, which is required for maximal export efficiency – triggering efficient capture or controlling deployment of nascent proteins (Fig. 1) [10]. Whether the human homologs of Erv14, CNIH1 through CNIH4, participate in similar facilitated export of CFTR remains to be determined. 3. CFTR membrane stability – a role for cAMP Once at the cell surface, CFTR stability is controlled by multiple protein interactors that regulate not only anterograde traffic to the cell surface, but also its endocytosis and recycling to achieve a fine and tight modulation of CFTR membrane levels. Among the different interactors, protein kinases have been identified as key regulators. Phosphorylation of CFTR at either its first nucleotide-binding domain by spleen tyrosine kinase (SYK) or at its regulatory domain by lemur tyrosine kinase 2 (LMTK2) regulate CFTR PM levels - phosphorylation by SYK leads to a decrease in CFTR at the PM, whereas LMTK2 inhibition or down-regulation stabilizes CFTR at the PM [11–13]. Major signaling pathways also have been implicated in the regulation of CFTR membrane stability including members of the Rho family of small GTPases – long reported as key regulators of the actin cytoskeleton remodelling. In particular, the small GTPase Rac1 was reported to regulate CFTR through a mechanism that promotes its anchoring to the cytoskeleton involving NHERF1 and ezrin [14]. Recent work reported that the second messenger cAMP also plays a role in regulating CFTR PM stability. The classical view according to which cAMP intracellular effects are exclusively mediated by cAMP-dependent PKA, which phosphorylates CFTR's R domain directly, was challenged by the discovery of a family of guanine nucleotide-exchange factors, called EPACs, that also respond to cAMP levels but do not lead to PKA activation [15]. EPACs regulate cell-to-cell and cell-to-matrix adhesion, cytoskeletal rearrangements, and cell polarization, processes which are dysregulated in CF [16]. In response to cAMP binding, EPACs shift to the PM anchoring through ezrin-radixin-moesin (ERM) proteins, previously known to link CFTR to the actin cytoskeleton and to facilitate cAMP-driven CFTR activation by tethering PKA
Fig. 1. Yor1 exit from the endoplasmic reticulum. Efficient capture into COPII vesicles relies on three interactions: (i) binding of the cargo adaptor, Sec24 to a sorting signal on the cargo, Yor1 (black star); (ii) Sec24 binding a sorting signal on the cargo receptor, Erv14, (green star); and (iii) interaction within the lipid bilayer between Erv14 and Yor1 (red star). This last interaction may be folding dependent such that only assembled transmembrane helices are recognized by Erv14. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.) (Adapted from [6,10].) Please cite this article as: Farinha CM, et al, Protein and lipid interactions – Modulating CFTR trafficking and rescue, J Cyst Fibros (2017), http://dx.doi.org/10.1016/ j.jcf.2017.08.014
C.M. Farinha et al. / Journal of Cystic Fibrosis xx (2017) xxx–xxx
in their close proximity. This suggests that activation of both CFTR and EPACs might be spatially and temporally coincident. Recent work has shown that CFTR interacts and co-localizes with Epac1 and that Epac1 activation stabilizes CFTR at the PM [17]. This process is dependent on the PDZ adaptor protein NHERF1 but not on ezrin [17]. These findings identify a novel CFTR interacting protein that links cAMP signaling to CFTR in a previously unreported mechanism. These data indicate that cAMP signaling regulates CFTR through two different but complementary pathways – PKA and EPAC1 (Fig. 2). This process involves a reshaping of CFTR interactions leading to a more robust anchoring to the cytoskeleton. 4. Membrane function – from internal to external regulators The ultimate goal of regulating CFTR PM levels is to allow its function as a chloride channel, through which anion secretion contributes to the hydration of epithelial surfaces. CFTR function is regulated by several key players with kinases once again having a prominent role [18]. This primarily involves PKA, but other kinases are known to phosphorylate CFTR leading to either its activation (e.g. cGMP-dependent protein kinase, Src and proline-rich tyrosine kinase Pyk2 [19,20]) or inhibition (e.g. AMP-dependent protein kinase - AMPK) [21], with some having a dual role in channel activity (e.g. PKA and protein kinase C - PKC [22,23]) depending on the consensus site phosphorylated [24,25]. Interactions at either the N-terminus or C-terminus of CFTR have also been reported to regulate channel activity, including PDZ proteins such as NHERF1 and CAL or
3
other membrane proteins that interact with CFTR through large protein complexes such as the adenosine 2b receptor or the beta-adrenergic receptor [26]. In addition to protein-mediated regulation, interaction with membrane lipids also seems to play a role in CFTR stability and function, although little is known about the full impact of lipids and lipid-mediated signaling pathways on CFTR. Interactions between CFTR and cholesterol have been described, and these interactions regulate the distribution and dynamics of CFTR in plasma membranes [27,28]; however, it is not clear whether interactions with cholesterol directly influence CFTR channel function. The impact of annular lipids, which comprise the bulk of membrane in which the protein sits, also has not been determined. However, a role for regulation of CFTR activity by sphingomyelin, and/or sphingomyelin-mediated signaling pathways, has been suggested previously [29,30]. Several types of bacteria that infect the lungs of CF patients, including Pseudomonas aeruginosa and Staphylococcus aureus, express sphingomyelinase C (SMase) as a virulence factor. SMase inhibits CFTR chloride channel activity in multiple cell systems, an effect that could exacerbate disease in CF and COPD patients [31]. Recent work has elucidated the mechanism through which sphingomyelin catalysis inhibits CFTR [32]. In Xenopus oocytes, it was shown that CFTR channels inhibited by SMase remained at the PM, suggesting the mechanism was tied to channel gating. Inhibition is state dependent, but occurs through a mechanism independent of the R domain. The sensitivity to the effect of SMase was changed under modulation of channel gating kinetics, by either introduction of
Fig. 2. CFTR stabilization by activation of EPAC1. Binding to NHERF1 anchors CFTR (via ezrin) to the actin cytoskeleton. In cells with low levels of cAMP near the PM, EPAC1 is inactive and its interaction with CFTR is not promoted. In cells with high levels of cAMP near the PM, both PKA and EPAC1 are active, promoting the opening of the channel and stabilizing CFTR at the PM, respectively. PDZ - PSD95, Dlg1, ZO-1 binding motif. (Adapted from [17].) Please cite this article as: Farinha CM, et al, Protein and lipid interactions – Modulating CFTR trafficking and rescue, J Cyst Fibros (2017), http://dx.doi.org/10.1016/ j.jcf.2017.08.014
4
C.M. Farinha et al. / Journal of Cystic Fibrosis xx (2017) xxx–xxx
the K1250A mutation or the treatment with VX-770, a CFTR potentiator – with these observations confirming that treatment with SMase influences channel gating. Some mutations that impede CFTR gating led to an increase in sensitivity suggesting that SMase targets a subset of closed states. Validation in primary bronchial epithelial cells showed that incubation with SMase, at the basolateral but not at the apical membrane, induced a change in sphingomyelin distribution as well as a decrease in CFTR currents stimulated by forskolin and VX-770. Overall, this work shows that SMase locks CFTR in a closed state, causing its inhibition, an effect that is also observed for endogenous CFTR expressed in HBE cells [32]. 5. Novel interactors – clues to therapy? Recent years have brought great advances in the therapy of CF. The identification and approval of modulators that correct the basic defect had significant impact in the life of CF patients [33,34], especially those carrying the mutations for which the existing therapies have been approved or are effective. However, the modest efficacy of the available therapies – at least in some patients – and the fact that a large number of mutations have not yet been included in this list still demand the search for better therapies. The need for a combinatorial approach to tackle most of the mutations – with the most evident example being F508del [35] – suggests that identification of novel mechanisms will probably give a valuable contribution to the scene. Identification of novel proteins involved in regulating CFTR exit from the ER, such as the ones described above [6] or elsewhere [36], or of mechanisms contributing to the stabilization of rescued mutant CFTR at the PM – still poorly explored as a therapeutic approach – will certainly have an impact on the advancement of alternative therapies. Characterization of mechanisms that modulate the efficacy of existing therapies may also have an impact. Bacterial burden directly influences the action of modulators, as shown previously for VX-809 [37] and strengthened by the recent observation that bacterial SMase also decreases the effect of the potentiator VX-770 [32]. Clearly, despite recent advances, there is still plenty of space for improvement so that better, alternative, novel therapies can be offered to every individual with CF. Acknowledgements Work supported by UID/MULTI/04046/2013 centre grant from FCT, Portugal (to BioISI), Gilead Génese PGG039-2014 and ERS Romain Pauwels Award (to CM Farinha), Medical Research Council MC_UP_1201/10 (to E Miller) and CFFdn MCCART15R0 (to N McCarty). References [1] Farinha CM, Matos P, Amaral MD. Control of CFTR membrane trafficking: not just from the ER to the Golgi. FEBS J 2013. [2] Roxo-Rosa M, Xu Z, Schmidt A, Neto M, Cai Z, Soares CM, et al. Revertant mutants G550E and 4RK rescue cystic fibrosis mutants in the first nucleotide-binding domain of CFTR by different mechanisms. Proc Natl Acad Sci U S A 2006;103:17891–6.
[3] Farinha CM, King-Underwood J, Sousa M, Correia AR, Henriques BJ, Roxo-Rosa M, et al. Revertants, low temperature, and correctors reveal the mechanism of F508del-CFTR rescue by VX-809 and suggest multiple agents for full correction. Chem Biol 2013;20:943–55. [4] Farinha CM, Amaral MD. Most F508del-CFTR is targeted to degradation at an early folding checkpoint and independently of calnexin. Mol Cell Biol 2005;25:5242–52. [5] Coppinger JA, Hutt DM, Razvi A, Koulov AV, Pankow S, Yates III JR, et al. A chaperone trap contributes to the onset of cystic fibrosis. PLoS One 2012;7:e37682. [6] Louie RJ, Guo J, Rodgers JW, White R, Shah N, Pagant S, et al. A yeast phenomic model for the gene interaction network modulating CFTRDeltaF508 protein biogenesis. Genome Med 2012;4:103. [7] Jonikas MC, Collins SR, Denic V, Oh E, Quan EM, Schmid V, et al. Comprehensive characterization of genes required for protein folding in the endoplasmic reticulum. Science 2009;323:1693–7. [8] Veit G, Oliver K, Apaja PM, Perdomo D, Bidaud-Meynard A, Lin ST, et al. Ribosomal stalk protein silencing partially corrects the DeltaF508-CFTR functional expression defect. PLoS Biol 2016;14:e1002462. [9] Wang X, Matteson J, An Y, Moyer B, Yoo JS, Bannykh S, et al. COPIIdependent export of cystic fibrosis transmembrane conductance regulator from the ER uses a di-acidic exit code. J Cell Biol 2004;167:65–74. [10] Pagant S, Wu A, Edwards S, Diehl F, Miller EA. Sec24 is a coincidence detector that simultaneously binds two signals to drive ER export. Curr Biol 2015;25:403–12. [11] Luz S, Cihil KM, Brautigan DL, Amaral MD, Farinha CM, SwiateckaUrban A. LMTK2 mediated phosphorylation regulates CFTR endocytosis in human airway epithelial cells. J Biol Chem 2014. [12] Luz S, Kongsuphol P, Mendes AI, Romeiras F, Sousa M, Schreiber R, et al. Contribution of casein kinase 2 and spleen tyrosine kinase to CFTR trafficking and protein kinase A-induced activity. Mol Cell Biol 2011;31: 4392–404. [13] Mendes AI, Matos P, Moniz S, Luz S, Amaral MD, Farinha CM, et al. Antagonistic regulation of CFTR cell surface expression by protein kinases WNK4 and spleen tyrosine kinase. Mol Cell Biol 2011. [14] Moniz S, Sousa M, Moraes BJ, Mendes AI, Palma M, Barreto C, et al. HGF stimulation of Rac1 signaling enhances pharmacological correction of the most prevalent cystic fibrosis mutant F508del-CFTR. ACS Chem Biol 2013;8:432–42. [15] Ponsioen B, Gloerich M, Ritsma L, Rehmann H, Bos JL, Jalink K. Direct spatial control of Epac1 by cyclic AMP. Mol Cell Biol 2009;29:2521–31. [16] Parnell E, Smith BO, Palmer TM, Terrin A, Zaccolo M, Yarwood SJ. Regulation of the inflammatory response of vascular endothelial cells by EPAC1. Br J Pharmacol 2012;166:434–46. [17] Lobo MJ, Amaral MD, Zaccolo M, Farinha CM. EPAC1 activation by cAMP stabilizes CFTR at the membrane by promoting its interaction with NHERF1. J Cell Sci 2016;129:2599–612. [18] Farinha CM, Swiatecka-Urban A, Brautigan DL, Jordan P. Regulatory crosstalk by protein kinases on CFTR trafficking and activity. Front Chem 2016;4:1. [19] Billet A, Jia Y, Jensen T, Riordan JR, Hanrahan JW. Regulation of the cystic fibrosis transmembrane conductance regulator anion channel by tyrosine phosphorylation. FASEB J 2015;29:3945–53. [20] Billet A, Jia Y, Jensen TJ, Hou YX, Chang XB, Riordan JR, et al. Potential sites of CFTR activation by tyrosine kinases. Channels (Austin) 2015;0. [21] Hallows KR, Raghuram V, Kemp BE, Witters LA, Foskett JK. Inhibition of cystic fibrosis transmembrane conductance regulator by novel interaction with the metabolic sensor AMP-activated protein kinase. J Clin Invest 2000; 105:1711–21. [22] Chappe V, Hinkson DA, Howell LD, Evagelidis A, Liao J, Chang XB, et al. Stimulatory and inhibitory protein kinase C consensus sequences regulate the cystic fibrosis transmembrane conductance regulator. Proc Natl Acad Sci U S A 2004;101:390–5. [23] Jia Y, Mathews CJ, Hanrahan JW. Phosphorylation by protein kinase C is required for acute activation of cystic fibrosis transmembrane conductance regulator by protein kinase A. J Biol Chem 1997;272:4978–84. [24] Alzamora R, King Jr JD, Hallows KR. CFTR regulation by phosphorylation. Methods Mol Biol 2011;741:471–88.
Please cite this article as: Farinha CM, et al, Protein and lipid interactions – Modulating CFTR trafficking and rescue, J Cyst Fibros (2017), http://dx.doi.org/10.1016/ j.jcf.2017.08.014
C.M. Farinha et al. / Journal of Cystic Fibrosis xx (2017) xxx–xxx [25] Berger HA, Travis SM, Welsh MJ. Regulation of the cystic fibrosis transmembrane conductance regulator Cl− channel by specific protein kinases and protein phosphatases. J Biol Chem 1993;268:2037–47. [26] Guggino WB, Stanton BA. New insights into cystic fibrosis: molecular switches that regulate CFTR. Nat Rev Mol Cell Biol 2006;7:426–36. [27] Cholon DM, O'Neal WK, Randell SH, Riordan JR, Gentzsch M. Modulation of endocytic trafficking and apical stability of CFTR in primary human airway epithelial cultures. Am J Physiol Lung Cell Mol Physiol 2010;298:L304–14. [28] Abu-Arish A, Pandzic E, Goepp J, Matthes E, Hanrahan JW, Wiseman PW. Cholesterol modulates CFTR confinement in the plasma membrane of primary epithelial cells. Biophys J 2015;109:85–94. [29] Ito Y, Sato S, Ohashi T, Nakayama S, Shimokata K, Kume H. Reduction of airway anion secretion via CFTR in sphingomyelin pathway. Biochem Biophys Res Commun 2004;324:901–8. [30] Ramu Y, Xu Y, Lu Z. Inhibition of CFTR Cl− channel function caused by enzymatic hydrolysis of sphingomyelin. Proc Natl Acad Sci U S A 2007; 104:6448–53. [31] Becker KA, Riethmuller J, Luth A, Doring G, Kleuser B, Gulbins E. Acid sphingomyelinase inhibitors normalize pulmonary ceramide and inflammation in cystic fibrosis. Am J Respir Cell Mol Biol 2010;42:716–24.
5
[32] Stauffer BB, Cui G, Cottrill KA, Infield DT, McCarty NA. Bacterial sphingomyelinase is a state-dependent inhibitor of the cystic fibrosis transmembrane conductance regulator (CFTR). Sci Rep 2017;7:2931. [33] Van Goor F, Hadida S, Grootenhuis PD, Burton B, Cao D, Neuberger T, et al. Rescue of CF airway epithelial cell function in vitro by a CFTR potentiator, VX-770. Proc Natl Acad Sci U S A 2009;106:18825–30. [34] Van Goor F, Hadida S, Grootenhuis PD, Burton B, Stack JH, Straley KS, et al. Correction of the F508del-CFTR protein processing defect in vitro by the investigational drug VX-809. Proc Natl Acad Sci U S A 2011;108: 18843–8. [35] Farinha CM, Matos P. Repairing the basic defect in cystic fibrosis - one approach is not enough. FEBS J 2016;283:246–64. [36] Botelho HM, Uliyakina I, Awatade NT, Proenca MC, Tischer C, Sirianant L, et al. Protein traffic disorders: an effective high-throughput fluorescence microscopy pipeline for drug discovery. Sci Rep 2015;5:9038. [37] Stanton BA, Coutermarsh B, Barnaby R, Hogan D. Pseudomonas aeruginosa reduces VX-809 stimulated F508del-CFTR chloride secretion by airway epithelial cells. PLoS One 2015;10:e0127742.
Please cite this article as: Farinha CM, et al, Protein and lipid interactions – Modulating CFTR trafficking and rescue, J Cyst Fibros (2017), http://dx.doi.org/10.1016/ j.jcf.2017.08.014
Journal of Cystic Fibrosis xx (2017) xxx – xxx www.elsevier.com/locate/jcf
Original Article
Protein and lipid interactions – Modulating CFTR trafficking and rescue Carlos M. Farinha a,⁎, Elizabeth Miller b , Nael McCarty c a
University of Lisboa, Faculty of Sciences, BioISI - Biosystems & Integrative Sciences Institute, Campo Grande, 1749-016 Lisboa, Portugal b MRC Laboratory of Molecular Biology, Cambridge, UK c Emory University School of Medicine and Children's Healthcare of Atlanta, Atlanta, GA, USA Received 8 July 2017; accepted 28 August 2017
Abstract Different levels of CFTR regulation in the cell contribute to a stringent control of chloride secretion in epithelia. Tuning of chloride transport is achieved by modulating CFTR biogenesis, exit from the endoplasmic reticulum, trafficking, membrane stability and channel activity. In this short review, we summarize recent findings identifying interactions with other proteins – directly or through membrane lipids – and briefly discuss how these observations can provide clues to the design of better therapeutic approaches. © 2017 Published by Elsevier B.V. on behalf of European Cystic Fibrosis Society. Keywords: Endoplasmic reticulum; Membrane stability; cAMP; Lipids; Sphingomyelin
1. Introduction Understanding CFTR folding, trafficking and function has been a matter of great priority in the search for improved therapeutic approaches for patients with CF. After co-translational insertion in the membrane of the endoplasmic reticulum (ER) and core-glycosylation, CFTR undergoes a complex succession of steps with the main goal of checking the overall quality of its conformation that will ultimately lead to ER exit and traffic through the secretory pathway [1–3]. Failure to pass the various checkpoints of ER quality control targets the most frequent disease-causing mutant protein (F508del-CFTR) for premature degradation. If folding assessment at the ER allows CFTR to traffic, the protein passes through the Golgi to finally reach the plasma membrane where its stability and function as a protein kinase A (PKA) phosphorylation-regulated chloride channel are finely tuned. Interactions with other proteins but also with membrane lipids are crucial in regulating CFTR's life cycle. This
⁎ Corresponding author at: Departamento de Química e Bioquímica, Faculdade de Ciências da Universidade de Lisboa, Campo Grande, 1749-016 Lisboa, Portugal. E-mail address: [email protected] (C.M. Farinha).
short review will highlight some novel topics on CFTR regulation by different interactions.
2. Gene interactions regulating CFTR exit from the ER Targeting of F508del-CFTR to degradation takes place at the ER as a result of an intricate series of events that constitute a quality control surveillance system within the secretory pathway. Several studies have characterized different checkpoints assessing CFTR biogenesis in the ER [1,2,4,5], but the mechanistic basis for this surveillance is still not fully understood. Recent work has used a systematic approach to discover proteins that influence the biogenesis of misfolded ABC transporters more broadly using yeast as a model organism and Yor1 as a model protein [6]. Yor1 acts at the plasma membrane as a drug pump to confer resistance to oligomycin and, as in CFTR, misfolding mutations cause ER retention and proteasomal degradation, leading to oligomycin sensitivity [6]. The F508del-CFTR equivalent is F670del-Yor1 that results in misfolding and reduced half-life. To gain insight into the gene interaction network that regulates ER exit, double mutants for Yor1 and 5000 other genes were assessed based on alteration in the dose response to growth inhibition by oligomycin
http://dx.doi.org/10.1016/j.jcf.2017.08.014 1569-1993© 2017 Published by Elsevier B.V. on behalf of European Cystic Fibrosis Society. Please cite this article as: Farinha CM, et al, Protein and lipid interactions – Modulating CFTR trafficking and rescue, J Cyst Fibros (2017), http://dx.doi.org/10.1016/ j.jcf.2017.08.014
2
C.M. Farinha et al. / Journal of Cystic Fibrosis xx (2017) xxx–xxx
(normally extruded by Yor1, a process defective in the mutant strain). Such approaches evidenced that yeast gene interactions affecting the biogenesis of F670del-Yor1 were representative of human homologs previously found to modulate the processing of F508del-CFTR in mammalian cells – including syntaxins, Rab proteins, and ERQC proteins [6]. Interestingly, this study determined that the biogenesis of such mutants is positively influenced by the ER membrane complex (EMC) [7], an observation that was validated biochemically by knocking down an EMC ortholog in a human cell line expressing F508del-CFTR. The EMC complex seems to be involved in the detection of protein misfolding at the earliest stages of protein synthesis. Such detection generates feedback to the ribosome that halts or slows translation, serving to either promote folding or prevent aberrant proteins from entering the secretory pathway. These observations are in agreement with data identifying a role for the ribosomal stalk protein (RPL) 12 in CFTR biogenesis. Originally identified in a yeast screen also using F670del-Yor1, RPL12 was shown to influence CFTR elongation rate, with its knock-down enabling partial rescue of F508del-CFTR mislocalization and ER retention [8]. Phenomic analysis using yeast models has also contributed to a further validation of the ER exit sites as an essential QC checkpoint for CFTR. Incorporation of this channel (as well as other secretory proteins) into ER-derived vesicles involves recognition of cytosolic signals by the COPII coat protein, Sec24. In CFTR, this process was previously shown to involve the recognition of a diacidic exit code DAD (at residues 565–567), whose abrogation impairs CFTR trafficking, without a major effect on its folding [3,9]. In addition to export motifs in cargo proteins, ER export receptors are also involved in regulating cargo diversity and recruitment into vesicles. Among these, Erv14 (ER-derived vesicle protein of 14 kDa) mediates export of transmembrane proteins despite their potential to directly interact with Sec24. Using a targeted mutagenesis approach, this protein was recently shown to interact with client proteins through conserved residues in its second transmembrane domain [10]. This interaction allows Erv14 to function as a canonical cargo receptor coupling membrane proteins, including the CFTR homolog Yor1, to the COPII component Sec24. Interestingly
however, such interaction seems to work on a dual mode, involving cargo and coat, which is required for maximal export efficiency – triggering efficient capture or controlling deployment of nascent proteins (Fig. 1) [10]. Whether the human homologs of Erv14, CNIH1 through CNIH4, participate in similar facilitated export of CFTR remains to be determined. 3. CFTR membrane stability – a role for cAMP Once at the cell surface, CFTR stability is controlled by multiple protein interactors that regulate not only anterograde traffic to the cell surface, but also its endocytosis and recycling to achieve a fine and tight modulation of CFTR membrane levels. Among the different interactors, protein kinases have been identified as key regulators. Phosphorylation of CFTR at either its first nucleotide-binding domain by spleen tyrosine kinase (SYK) or at its regulatory domain by lemur tyrosine kinase 2 (LMTK2) regulate CFTR PM levels - phosphorylation by SYK leads to a decrease in CFTR at the PM, whereas LMTK2 inhibition or down-regulation stabilizes CFTR at the PM [11–13]. Major signaling pathways also have been implicated in the regulation of CFTR membrane stability including members of the Rho family of small GTPases – long reported as key regulators of the actin cytoskeleton remodelling. In particular, the small GTPase Rac1 was reported to regulate CFTR through a mechanism that promotes its anchoring to the cytoskeleton involving NHERF1 and ezrin [14]. Recent work reported that the second messenger cAMP also plays a role in regulating CFTR PM stability. The classical view according to which cAMP intracellular effects are exclusively mediated by cAMP-dependent PKA, which phosphorylates CFTR's R domain directly, was challenged by the discovery of a family of guanine nucleotide-exchange factors, called EPACs, that also respond to cAMP levels but do not lead to PKA activation [15]. EPACs regulate cell-to-cell and cell-to-matrix adhesion, cytoskeletal rearrangements, and cell polarization, processes which are dysregulated in CF [16]. In response to cAMP binding, EPACs shift to the PM anchoring through ezrin-radixin-moesin (ERM) proteins, previously known to link CFTR to the actin cytoskeleton and to facilitate cAMP-driven CFTR activation by tethering PKA
Fig. 1. Yor1 exit from the endoplasmic reticulum. Efficient capture into COPII vesicles relies on three interactions: (i) binding of the cargo adaptor, Sec24 to a sorting signal on the cargo, Yor1 (black star); (ii) Sec24 binding a sorting signal on the cargo receptor, Erv14, (green star); and (iii) interaction within the lipid bilayer between Erv14 and Yor1 (red star). This last interaction may be folding dependent such that only assembled transmembrane helices are recognized by Erv14. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.) (Adapted from [6,10].) Please cite this article as: Farinha CM, et al, Protein and lipid interactions – Modulating CFTR trafficking and rescue, J Cyst Fibros (2017), http://dx.doi.org/10.1016/ j.jcf.2017.08.014
C.M. Farinha et al. / Journal of Cystic Fibrosis xx (2017) xxx–xxx
in their close proximity. This suggests that activation of both CFTR and EPACs might be spatially and temporally coincident. Recent work has shown that CFTR interacts and co-localizes with Epac1 and that Epac1 activation stabilizes CFTR at the PM [17]. This process is dependent on the PDZ adaptor protein NHERF1 but not on ezrin [17]. These findings identify a novel CFTR interacting protein that links cAMP signaling to CFTR in a previously unreported mechanism. These data indicate that cAMP signaling regulates CFTR through two different but complementary pathways – PKA and EPAC1 (Fig. 2). This process involves a reshaping of CFTR interactions leading to a more robust anchoring to the cytoskeleton. 4. Membrane function – from internal to external regulators The ultimate goal of regulating CFTR PM levels is to allow its function as a chloride channel, through which anion secretion contributes to the hydration of epithelial surfaces. CFTR function is regulated by several key players with kinases once again having a prominent role [18]. This primarily involves PKA, but other kinases are known to phosphorylate CFTR leading to either its activation (e.g. cGMP-dependent protein kinase, Src and proline-rich tyrosine kinase Pyk2 [19,20]) or inhibition (e.g. AMP-dependent protein kinase - AMPK) [21], with some having a dual role in channel activity (e.g. PKA and protein kinase C - PKC [22,23]) depending on the consensus site phosphorylated [24,25]. Interactions at either the N-terminus or C-terminus of CFTR have also been reported to regulate channel activity, including PDZ proteins such as NHERF1 and CAL or
3
other membrane proteins that interact with CFTR through large protein complexes such as the adenosine 2b receptor or the beta-adrenergic receptor [26]. In addition to protein-mediated regulation, interaction with membrane lipids also seems to play a role in CFTR stability and function, although little is known about the full impact of lipids and lipid-mediated signaling pathways on CFTR. Interactions between CFTR and cholesterol have been described, and these interactions regulate the distribution and dynamics of CFTR in plasma membranes [27,28]; however, it is not clear whether interactions with cholesterol directly influence CFTR channel function. The impact of annular lipids, which comprise the bulk of membrane in which the protein sits, also has not been determined. However, a role for regulation of CFTR activity by sphingomyelin, and/or sphingomyelin-mediated signaling pathways, has been suggested previously [29,30]. Several types of bacteria that infect the lungs of CF patients, including Pseudomonas aeruginosa and Staphylococcus aureus, express sphingomyelinase C (SMase) as a virulence factor. SMase inhibits CFTR chloride channel activity in multiple cell systems, an effect that could exacerbate disease in CF and COPD patients [31]. Recent work has elucidated the mechanism through which sphingomyelin catalysis inhibits CFTR [32]. In Xenopus oocytes, it was shown that CFTR channels inhibited by SMase remained at the PM, suggesting the mechanism was tied to channel gating. Inhibition is state dependent, but occurs through a mechanism independent of the R domain. The sensitivity to the effect of SMase was changed under modulation of channel gating kinetics, by either introduction of
Fig. 2. CFTR stabilization by activation of EPAC1. Binding to NHERF1 anchors CFTR (via ezrin) to the actin cytoskeleton. In cells with low levels of cAMP near the PM, EPAC1 is inactive and its interaction with CFTR is not promoted. In cells with high levels of cAMP near the PM, both PKA and EPAC1 are active, promoting the opening of the channel and stabilizing CFTR at the PM, respectively. PDZ - PSD95, Dlg1, ZO-1 binding motif. (Adapted from [17].) Please cite this article as: Farinha CM, et al, Protein and lipid interactions – Modulating CFTR trafficking and rescue, J Cyst Fibros (2017), http://dx.doi.org/10.1016/ j.jcf.2017.08.014
4
C.M. Farinha et al. / Journal of Cystic Fibrosis xx (2017) xxx–xxx
the K1250A mutation or the treatment with VX-770, a CFTR potentiator – with these observations confirming that treatment with SMase influences channel gating. Some mutations that impede CFTR gating led to an increase in sensitivity suggesting that SMase targets a subset of closed states. Validation in primary bronchial epithelial cells showed that incubation with SMase, at the basolateral but not at the apical membrane, induced a change in sphingomyelin distribution as well as a decrease in CFTR currents stimulated by forskolin and VX-770. Overall, this work shows that SMase locks CFTR in a closed state, causing its inhibition, an effect that is also observed for endogenous CFTR expressed in HBE cells [32]. 5. Novel interactors – clues to therapy? Recent years have brought great advances in the therapy of CF. The identification and approval of modulators that correct the basic defect had significant impact in the life of CF patients [33,34], especially those carrying the mutations for which the existing therapies have been approved or are effective. However, the modest efficacy of the available therapies – at least in some patients – and the fact that a large number of mutations have not yet been included in this list still demand the search for better therapies. The need for a combinatorial approach to tackle most of the mutations – with the most evident example being F508del [35] – suggests that identification of novel mechanisms will probably give a valuable contribution to the scene. Identification of novel proteins involved in regulating CFTR exit from the ER, such as the ones described above [6] or elsewhere [36], or of mechanisms contributing to the stabilization of rescued mutant CFTR at the PM – still poorly explored as a therapeutic approach – will certainly have an impact on the advancement of alternative therapies. Characterization of mechanisms that modulate the efficacy of existing therapies may also have an impact. Bacterial burden directly influences the action of modulators, as shown previously for VX-809 [37] and strengthened by the recent observation that bacterial SMase also decreases the effect of the potentiator VX-770 [32]. Clearly, despite recent advances, there is still plenty of space for improvement so that better, alternative, novel therapies can be offered to every individual with CF. Acknowledgements Work supported by UID/MULTI/04046/2013 centre grant from FCT, Portugal (to BioISI), Gilead Génese PGG039-2014 and ERS Romain Pauwels Award (to CM Farinha), Medical Research Council MC_UP_1201/10 (to E Miller) and CFFdn MCCART15R0 (to N McCarty). References [1] Farinha CM, Matos P, Amaral MD. Control of CFTR membrane trafficking: not just from the ER to the Golgi. FEBS J 2013. [2] Roxo-Rosa M, Xu Z, Schmidt A, Neto M, Cai Z, Soares CM, et al. Revertant mutants G550E and 4RK rescue cystic fibrosis mutants in the first nucleotide-binding domain of CFTR by different mechanisms. Proc Natl Acad Sci U S A 2006;103:17891–6.
[3] Farinha CM, King-Underwood J, Sousa M, Correia AR, Henriques BJ, Roxo-Rosa M, et al. Revertants, low temperature, and correctors reveal the mechanism of F508del-CFTR rescue by VX-809 and suggest multiple agents for full correction. Chem Biol 2013;20:943–55. [4] Farinha CM, Amaral MD. Most F508del-CFTR is targeted to degradation at an early folding checkpoint and independently of calnexin. Mol Cell Biol 2005;25:5242–52. [5] Coppinger JA, Hutt DM, Razvi A, Koulov AV, Pankow S, Yates III JR, et al. A chaperone trap contributes to the onset of cystic fibrosis. PLoS One 2012;7:e37682. [6] Louie RJ, Guo J, Rodgers JW, White R, Shah N, Pagant S, et al. A yeast phenomic model for the gene interaction network modulating CFTRDeltaF508 protein biogenesis. Genome Med 2012;4:103. [7] Jonikas MC, Collins SR, Denic V, Oh E, Quan EM, Schmid V, et al. Comprehensive characterization of genes required for protein folding in the endoplasmic reticulum. Science 2009;323:1693–7. [8] Veit G, Oliver K, Apaja PM, Perdomo D, Bidaud-Meynard A, Lin ST, et al. Ribosomal stalk protein silencing partially corrects the DeltaF508-CFTR functional expression defect. PLoS Biol 2016;14:e1002462. [9] Wang X, Matteson J, An Y, Moyer B, Yoo JS, Bannykh S, et al. COPIIdependent export of cystic fibrosis transmembrane conductance regulator from the ER uses a di-acidic exit code. J Cell Biol 2004;167:65–74. [10] Pagant S, Wu A, Edwards S, Diehl F, Miller EA. Sec24 is a coincidence detector that simultaneously binds two signals to drive ER export. Curr Biol 2015;25:403–12. [11] Luz S, Cihil KM, Brautigan DL, Amaral MD, Farinha CM, SwiateckaUrban A. LMTK2 mediated phosphorylation regulates CFTR endocytosis in human airway epithelial cells. J Biol Chem 2014. [12] Luz S, Kongsuphol P, Mendes AI, Romeiras F, Sousa M, Schreiber R, et al. Contribution of casein kinase 2 and spleen tyrosine kinase to CFTR trafficking and protein kinase A-induced activity. Mol Cell Biol 2011;31: 4392–404. [13] Mendes AI, Matos P, Moniz S, Luz S, Amaral MD, Farinha CM, et al. Antagonistic regulation of CFTR cell surface expression by protein kinases WNK4 and spleen tyrosine kinase. Mol Cell Biol 2011. [14] Moniz S, Sousa M, Moraes BJ, Mendes AI, Palma M, Barreto C, et al. HGF stimulation of Rac1 signaling enhances pharmacological correction of the most prevalent cystic fibrosis mutant F508del-CFTR. ACS Chem Biol 2013;8:432–42. [15] Ponsioen B, Gloerich M, Ritsma L, Rehmann H, Bos JL, Jalink K. Direct spatial control of Epac1 by cyclic AMP. Mol Cell Biol 2009;29:2521–31. [16] Parnell E, Smith BO, Palmer TM, Terrin A, Zaccolo M, Yarwood SJ. Regulation of the inflammatory response of vascular endothelial cells by EPAC1. Br J Pharmacol 2012;166:434–46. [17] Lobo MJ, Amaral MD, Zaccolo M, Farinha CM. EPAC1 activation by cAMP stabilizes CFTR at the membrane by promoting its interaction with NHERF1. J Cell Sci 2016;129:2599–612. [18] Farinha CM, Swiatecka-Urban A, Brautigan DL, Jordan P. Regulatory crosstalk by protein kinases on CFTR trafficking and activity. Front Chem 2016;4:1. [19] Billet A, Jia Y, Jensen T, Riordan JR, Hanrahan JW. Regulation of the cystic fibrosis transmembrane conductance regulator anion channel by tyrosine phosphorylation. FASEB J 2015;29:3945–53. [20] Billet A, Jia Y, Jensen TJ, Hou YX, Chang XB, Riordan JR, et al. Potential sites of CFTR activation by tyrosine kinases. Channels (Austin) 2015;0. [21] Hallows KR, Raghuram V, Kemp BE, Witters LA, Foskett JK. Inhibition of cystic fibrosis transmembrane conductance regulator by novel interaction with the metabolic sensor AMP-activated protein kinase. J Clin Invest 2000; 105:1711–21. [22] Chappe V, Hinkson DA, Howell LD, Evagelidis A, Liao J, Chang XB, et al. Stimulatory and inhibitory protein kinase C consensus sequences regulate the cystic fibrosis transmembrane conductance regulator. Proc Natl Acad Sci U S A 2004;101:390–5. [23] Jia Y, Mathews CJ, Hanrahan JW. Phosphorylation by protein kinase C is required for acute activation of cystic fibrosis transmembrane conductance regulator by protein kinase A. J Biol Chem 1997;272:4978–84. [24] Alzamora R, King Jr JD, Hallows KR. CFTR regulation by phosphorylation. Methods Mol Biol 2011;741:471–88.
Please cite this article as: Farinha CM, et al, Protein and lipid interactions – Modulating CFTR trafficking and rescue, J Cyst Fibros (2017), http://dx.doi.org/10.1016/ j.jcf.2017.08.014
C.M. Farinha et al. / Journal of Cystic Fibrosis xx (2017) xxx–xxx [25] Berger HA, Travis SM, Welsh MJ. Regulation of the cystic fibrosis transmembrane conductance regulator Cl− channel by specific protein kinases and protein phosphatases. J Biol Chem 1993;268:2037–47. [26] Guggino WB, Stanton BA. New insights into cystic fibrosis: molecular switches that regulate CFTR. Nat Rev Mol Cell Biol 2006;7:426–36. [27] Cholon DM, O'Neal WK, Randell SH, Riordan JR, Gentzsch M. Modulation of endocytic trafficking and apical stability of CFTR in primary human airway epithelial cultures. Am J Physiol Lung Cell Mol Physiol 2010;298:L304–14. [28] Abu-Arish A, Pandzic E, Goepp J, Matthes E, Hanrahan JW, Wiseman PW. Cholesterol modulates CFTR confinement in the plasma membrane of primary epithelial cells. Biophys J 2015;109:85–94. [29] Ito Y, Sato S, Ohashi T, Nakayama S, Shimokata K, Kume H. Reduction of airway anion secretion via CFTR in sphingomyelin pathway. Biochem Biophys Res Commun 2004;324:901–8. [30] Ramu Y, Xu Y, Lu Z. Inhibition of CFTR Cl− channel function caused by enzymatic hydrolysis of sphingomyelin. Proc Natl Acad Sci U S A 2007; 104:6448–53. [31] Becker KA, Riethmuller J, Luth A, Doring G, Kleuser B, Gulbins E. Acid sphingomyelinase inhibitors normalize pulmonary ceramide and inflammation in cystic fibrosis. Am J Respir Cell Mol Biol 2010;42:716–24.
5
[32] Stauffer BB, Cui G, Cottrill KA, Infield DT, McCarty NA. Bacterial sphingomyelinase is a state-dependent inhibitor of the cystic fibrosis transmembrane conductance regulator (CFTR). Sci Rep 2017;7:2931. [33] Van Goor F, Hadida S, Grootenhuis PD, Burton B, Cao D, Neuberger T, et al. Rescue of CF airway epithelial cell function in vitro by a CFTR potentiator, VX-770. Proc Natl Acad Sci U S A 2009;106:18825–30. [34] Van Goor F, Hadida S, Grootenhuis PD, Burton B, Stack JH, Straley KS, et al. Correction of the F508del-CFTR protein processing defect in vitro by the investigational drug VX-809. Proc Natl Acad Sci U S A 2011;108: 18843–8. [35] Farinha CM, Matos P. Repairing the basic defect in cystic fibrosis - one approach is not enough. FEBS J 2016;283:246–64. [36] Botelho HM, Uliyakina I, Awatade NT, Proenca MC, Tischer C, Sirianant L, et al. Protein traffic disorders: an effective high-throughput fluorescence microscopy pipeline for drug discovery. Sci Rep 2015;5:9038. [37] Stanton BA, Coutermarsh B, Barnaby R, Hogan D. Pseudomonas aeruginosa reduces VX-809 stimulated F508del-CFTR chloride secretion by airway epithelial cells. PLoS One 2015;10:e0127742.
Please cite this article as: Farinha CM, et al, Protein and lipid interactions – Modulating CFTR trafficking and rescue, J Cyst Fibros (2017), http://dx.doi.org/10.1016/ j.jcf.2017.08.014