cystic fibrosis and protein synthesis case study

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Case Study: Cystic Fibrosis Mutations

This case study is a follow-up to the Cystic Fibrosis Case Study where students explore how changes in transport proteins affects the movement of ions, resulting in a build-up of chloride ions and the symptoms of the disease.

Students were introduced to the idea that different mutations can cause differences in the transport proteins, but in the first version, the origin of these mutations was not discussed.

Eventually, students get to the chapter on DNA, RNA, and protein synthesis, so it’s a good time to circle back to the CF case and explore how mutations in DNA can affect the protein made by the ribosomes.

Students should already have some background in the central dogma, but a review may be in order to remind students how to transcribe DNA to RNA and then use a codon chart to determine the sequence of amino acids. This practice worksheet on using codon charts is something they may have done in freshman biology.

CFTR Mutations

This case explore frameshift mutations, missense mutations, and nonsense mutations. Students are given a section of DNA to transcribe and compare it to mutant DNA. Students should see that changes in DNA can result in changes in the synthesized protein, though some changes are more profound than others.

The link below is a Google Doc designed for remote learning but will work for in-class lessons. An original in-class version is also available, where it doesn’t have the colored text boxes.

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Shannan Muskopf

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Case Study: Cystic Fibrosis - CER

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This page is a draft and is under active development. 

Part I: A​ ​Case​ ​of​ ​Cystic​ ​Fibrosis

Dr. Weyland examined a six month old infant that had been admitted to University Hospital earlier in the day. The baby's parents had brought young Zoey to the emergency room because she had been suffering from a chronic cough. In addition, they said that Zoey sometimes would "wheeze" a lot more than they thought was normal for a child with a cold. Upon arriving at the emergency room, the attending pediatrician noted that salt crystals were present on Zoey's skin and called Dr. Weyland, a pediatric pulmonologist. Dr. Weyland suspects that baby Zoey may be suffering from cystic fibrosis.

CF affects more than 30,000 kids and young adults in the United States. It disrupts the normal function of epithelial cells — cells that make up the sweat glands in the skin and that also line passageways inside the lungs, pancreas, and digestive and reproductive systems.

The inherited CF gene directs the body's epithelial cells to produce a defective form of a protein called CFTR (or cystic fibrosis transmembrane conductance regulator) found in cells that line the lungs, digestive tract, sweat glands, and genitourinary system.

When the CFTR protein is defective, epithelial cells can't regulate the way that chloride ions pass across cell membranes. This disrupts the balance of salt and water needed to maintain a normal thin coating of mucus inside the lungs and other passageways. The mucus becomes thick, sticky, and hard to move, and can result in infections from bacterial colonization.

• "Woe to that child which when kissed on the forehead tastes salty. He is bewitched and soon will die" This is an old saying from the eighteenth century and describes one of the symptoms of CF (salty skin). Why do you think babies in the modern age have a better chance of survival than babies in the 18th century?
• What symptoms lead Dr. Weyland to his initial diagnosis?
• Consider the graph of infections, which organism stays relatively constant in numbers over a lifetime. What organism is most likely affecting baby Zoey?
• What do you think is the most dangerous time period for a patient with CF? Justify your answer.

Part​ ​II:​ ​ ​CF​ ​is​ ​a​ ​disorder​ ​of​ ​the​ ​cell​ ​membrane.

Imagine a door with key and combination locks on both sides, back and front. Now imagine trying to unlock that door blind-folded. This is the challenge faced by David Gadsby, Ph.D., who for years struggled to understand the highly intricate and unusual cystic fibrosis chloride channel – a cellular doorway for salt ions that is defective in people with cystic fibrosis.

His findings, reported in a series of three recent papers in the Journal of General Physiology, detail the type and order of molecular events required to open and close the gates of the cystic fibrosis chloride channel, or as scientists call it, the cystic fibrosis transmembrane conductance regulator (CFTR).

Ultimately, the research may have medical applications, though ironically not likely for most cystic fibrosis patients. Because two-thirds of cystic fibrosis patients fail to produce the cystic fibrosis channel altogether, a cure for most is expected to result from research focused on replacing the lost channel.

5. Suggest a molecular fix for a mutated CFTR channel. How would you correct it if you had the ability to tinker with it on a molecular level?

6. Why would treatment that targets the CFTR channel not be effective for 2⁄3 of those with cystic fibrosis?

7. Sweat glands cool the body by releasing perspiration (sweat) from the lower layers of the skin onto the surface. Sodium and chloride (salt) help carry water to the skin's surface and are then reabsorbed into the body. Why does a person with cystic fibrosis have salty tasting skin?

Part​ ​III:​ ​No​ ​cell​ ​is​ ​an​ ​island

Like people, cells need to communicate and interact with their environment to survive. One way they go about this is through pores in their outer membranes, called ion channels, which provide charged ions, such as chloride or potassium, with their own personalized cellular doorways. But, ion channels are not like open doors; instead, they are more like gateways with high-security locks that are opened and closed to carefully control the passage of their respective ions.

In the case of CFTR, chloride ions travel in and out of the cell through the channel’s guarded pore as a means to control the flow of water in and out of cells. In cystic fibrosis patients, this delicate salt/water balance is disturbed, most prominently in the lungs, resulting in thick coats of mucus that eventually spur life-threatening infections. Shown below are several mutations linked to CFTR:

8. Which mutation do you think would be easiest to correct. Justify your answer. 9. Consider what you know about proteins, why does the “folding” of the protein matter?

Part​ ​IV:​ ​Open​ ​sesame

Among the numerous ion channels in cell membranes, there are two principal types: voltage-gated and ligand-gated. Voltage-gated channels are triggered to open and shut their doors by changes in the electric potential difference across the membrane. Ligand-gated channels, in contrast, require a special “key” to unlock their doors, which usually comes in the form of a small molecule.

CFTR is a ligand-gated channel, but it’s an unusual one. Its “key” is ATP, a small molecule that plays a critical role in the storage and release of energy within cells in the body. In addition to binding the ATP, the CFTR channel must snip a phosphate group – one of three “P’s” – off the ATP molecule to function. But when, where and how often this crucial event takes place has remains obscure.

10. Compare the action of the ligand-gated channel to how an enzyme works.

11. Consider the model of the membrane channel, What could go wrong to prevent the channel from opening?

12. Where is ATP generated in the cell? How might ATP production affect the symptoms of cystic fibrosis?

13. Label the image below to show how the ligand-gated channel for CFTR works. Include a summary.

Part​ ​V:​ Can​ ​a​ ​Drug​ ​Treat​ ​Zoey’s​ ​Condition?

Dr. Weyland confirmed that Zoey does have cystic fibrosis and called the parents in to talk about potential treatments. “Good news, there are two experimental drugs that have shown promise in CF patients. These drugs can help Zoey clear the mucus from his lungs. Unfortunately, the drugs do not work in all cases.” The doctor gave the parents literature about the drugs and asked them to consider signing Zoey up for trials.

The​ ​Experimental​ ​Drugs

Ivacaftor TM is a potentiator that increases CFTR channel opening time. We know from the cell culture studies that this increases chloride transport by as much as 50% from baseline and restores it closer to what we would expect to observe in wild type CFTR. Basically, the drug increases CFTR activity by unlocking the gate that allows for the normal flow of salt and fluids.

In early trials, 144 patients all of whom were age over the age of 12 were treated with 150 mg of Ivacaftor twice daily. The total length of treatment was 48 weeks. Graph A shows changes in FEV (forced expiratory volume) with individuals using the drug versus a placebo. Graph B shows concentrations of chloride in patient’s sweat.

14. What is FEV? Describe a way that a doctor could take a measurement of FEV.

15. Why do you think it was important to have placebos in both of these studies?

16. Which graph do you think provides the most compelling evidence for the effectiveness of Ivacafor? Defend your choice.

17. Take a look at the mutations that can occur in the cell membrane proteins from Part III. For which mutation do you think Ivacaftor will be most effective? Justify your answer.

18. Would you sign Zoey up for clinical trials based on the evidence? What concerns would a parent have before considering an experimental drug?

Part​ ​VI:​ ​Zoey’s​ ​Mutation

Dr. Weyland calls a week later to inform the parents that genetic tests show that Zoey chromosomes show that she has two copies of the F508del mutation. This mutation, while the most common type of CF mutation, is also one that is difficult to treat with just Ivacaftor. There are still some options for treatment.

In people with the most common CF mutation, F508del, a series of problems prevents the CFTR protein from taking its correct shape and reaching its proper place on the cell surface. The cell recognizes the protein as not normal and targets it for degradation before it makes it to the cell surface. In order to treat this problem, we need to do two things: first, an agent to get the protein to the surface, and then ivacaftor (VX-770) to open up the channel and increase chloride transport. VX-809 has been identified as a way to help with the trafficking of the protein to the cell surface. When added VX-809 is added to ivacaftor (now called Lumacaftor,) the protein gets to the surface and also increases in chloride transport by increasing channel opening time.

In early trials, experiments were done in-vitro, where studies were done on cell cultures to see if the drugs would affect the proteins made by the cell. General observations can be made from the cells, but drugs may not work on an individual’s phenotype. A new type of research uses ex-vivo experiments, where rectal organoids (mini-guts) were grown from rectal biopsies of the patient that would be treated with the drug. Ex-vivo experiments are personalized medicine, each person may have different correctors and potentiators evaluated using their own rectal organoids. The graph below shows how each drug works for 8 different patients (#1-#8)

19. Compare ex-vivo trials to in-vitro trials.

20. One the graph, label the group that represents Ivacaftor and Lumacaftor. What is the difference between these two drugs?

21. Complete a CER Chart. If the profile labeled #7 is Zoey, rank the possible drug treatments in order of their effectiveness for her mutation. This is your CLAIM. Provide EVIDENCE​ to support your claim. Provide REASONING​ that explains why this treatment would be more effective than other treatments and why what works for Zoey may not work for other patients. This is where you tie the graph above to everything you have learned in this case. Attach a page.

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• J Biol Chem
• v.285(46); 2010 Nov 12

The Cystic Fibrosis-causing Mutation ΔF508 Affects Multiple Steps in Cystic Fibrosis Transmembrane Conductance Regulator Biogenesis *

Patrick h. thibodeau.

From the ‡ Department of Physiology and

John M. Richardson, III

the § Department of Physiology and Biophysics, The University of Alabama at Birmingham, Birmingham, Alabama 35294,

Linda Millen

Jarod watson, juan l. mendoza.

¶ Molecular Biophysics Graduate Program, The University of Texas Southwestern Medical Center, Dallas, Texas 75390,

the ‖ Department of Physiology, McGill University, Montreal, Quebec H3G 1Y6, Canada, and

Sharon Fischman

** Epix Pharmaceuticals, Lexington, Massachusetts 02421-3112

Hanoch Senderowitz

Gergely l. lukacs, philip j. thomas.

The deletion of phenylalanine 508 in the first nucleotide binding domain of the cystic fibrosis transmembrane conductance regulator is directly associated with >90% of cystic fibrosis cases. This mutant protein fails to traffic out of the endoplasmic reticulum and is subsequently degraded by the proteasome. The effects of this mutation may be partially reversed by the application of exogenous osmolytes, expression at low temperature, and the introduction of second site suppressor mutations. However, the specific steps of folding and assembly of full-length cystic fibrosis transmembrane conductance regulator (CFTR) directly altered by the disease-causing mutation are unclear. To elucidate the effects of the ΔF508 mutation, on various steps in CFTR folding, a series of misfolding and suppressor mutations in the nucleotide binding and transmembrane domains were evaluated for effects on the folding and maturation of the protein. The results indicate that the isolated NBD1 responds to both the ΔF508 mutation and intradomain suppressors of this mutation. In addition, identification of a novel second site suppressor of the defect within the second transmembrane domain suggests that ΔF508 also effects interdomain interactions critical for later steps in the biosynthesis of CFTR.

Introduction

The maturation of polytopic multidomain membrane proteins is a complex process that requires the proper folding and assembly of individual domains to form a functional complex ( 1 ). These processes may be tightly coupled and occur simultaneously or may proceed in a hierarchical fashion. In addition, these processes may proceed in either a co- or post-translational manner ( 2 , 3 ). The unique nature of these proteins often requires chaperone systems to promote the proper interactions both within and across multiple protein domains. Perturbations that alter the structures of the individual domains or that alter the interactions of these multi-domain complexes are recognized by the cellular quality control (QC) machines, which ultimately target the newly synthesized protein for maturation or degradation.

Studies of the cystic fibrosis transmembrane conductance regulator (CFTR), 5 the protein whose loss results in cystic fibrosis (CF) have provided insight into the folding of polytopic membrane proteins ( 4 ). CFTR is a member of the ABC-transporter family of proteins and is composed of five distinct domains; two transmembrane domains, TMD1 and TMD2; two nucleotide binding domains, NBD1 and NBD2; and a regulatory domain, R ( 4 ). The most common CF-causing mutation, the deletion of phenylalanine 508 (ΔF508), is located in the N-terminal cytoplasmic NBD1 ( 5 , – 9 ). This single amino acid deletion results in a dramatic reduction of mature, plasma membrane resident CFTR. The immature protein is arrested in an intermediate conformational state that is recognized by the cellular quality control machinery and targeted for degradation by the ubiquitin-proteasome system ( 10 , – 13 ). Previous work has shown that the ΔF508 CFTR can be “rescued” by a variety of treatments; that is, low temperature protein expression, the addition of osmolytes and chemical chaperones to cell culture medium, alterations to cellular quality control systems, and by additional mutations within NBD1 ( 14 , – 17 ).

Although most manipulations that rescue ΔF508 CFTR are likely nonspecific, mediated through gross changes to protein-protein and/or protein-solvent interactions, the identification of suppressor mutations indicates that the specific rescue of this folding defect is possible. A single mutation, R553Q, was first identified in a patient homozygous for the ΔF508 allele but having only a mild CF phenotype ( 18 ). Subsequently, in a screen for suppressor mutations of the ΔF508 defect, the original R553Q suppressor mutation was identified as were I539T, G550E, R553Q, and R555K ( 19 , – 21 ). When introduced into a ΔF508 background these mutations promoted trafficking and partially restored ΔF508 function at the plasma membrane.

As the ΔF508 and suppressor mutations are located within NBD1, they may alter the biochemical properties of the NBD ( i.e. folding efficiency or stability). Alternatively, they may alter the interaction of CFTR domains while leaving the biochemical and biophysical properties of the isolated NBD unaltered. The suppressors might also have little influence on the properties of the CFTR polypeptide in cis but may alter the interaction of cellular quality control machinery with CFTR, thereby promoting CFTR trafficking in trans ( 22 , 23 ). Finally, suppression of the ΔF508 defect may be the result of a combination of effects on specific intradomain, interdomain, and cellular components interactions.

High and low resolution structural information is available for the CFTR NBDs and homologous ABC -transporters, providing insight into the putative structure and association of CFTR domains. Structures of homologous bacterial transporter systems suggest that the Phe-508 position lies at the interface between the NBD and the fourth intracellular loop (ICL4) of TMD2 ( 7 , – 9 , 24 ). This interface is predicted to couple the energy of ATP-binding and hydrolysis in the NBDs to the transport or channel activity of the TMDs and provide specificity for the TMD-NBD interaction ( 25 ).

The structures of CFTR NBD1 show that the Phe-508 side chain is surface-exposed in the isolated domain. The chemical and physical character of this position contributes directly to the characteristics of the putative TMD-NBD domain-domain interaction surface ( 5 , 6 , 26 ). Consistent with the relatively high surface exposure of the 508 side chain, NBD1 tolerates several non-conservative missense mutations with minimal structural changes, although full-length CFTR fails to fold when charged, and bulky substitutions are made for the Phe-508 side chain ( 12 , 26 ).

Structures of ΔF508 NBD1 have been solved ( 6 , 27 ). Minimal changes to the protein backbone are evident, although local perturbations to the putative domain-domain interaction surface proximal to the Phe-508 position are seen ( 27 , 28 ). The alterations noted in the static structures of the missense and ΔF508 NBDs and the sensitivity of full-length CFTR to charged and bulky substitutions at the 508 position underlies models wherein appropriate NBD-TMD associations are altered. The structures of ΔF508 NBD1 solved to date include additional mutations introduced to increase soluble protein production and facilitate crystallization. These include known second-site suppressors of the ΔF508 mutation and novel solubilizing mutations ( 6 , 27 ).

The introduction of these additional mutations partially rescues the folding, trafficking, and function of ΔF508 CFTR ( 29 ). However, these mutations are not proximal to Phe-508 nor do they contribute directly to the TMD-NBD surface defined by the Phe-508 side chain ( 29 ). This suggests that alterations to the TMD-NBD surface of NBD1, as seen statically in the NBD1 crystal structures, are not the sole defect in ΔF508 CFTR maturation ( 19 , – 21 ).

Previous biochemical and biophysical studies have demonstrated that the properties of NBD1 are directly altered by the introduction of the ΔF508 mutation. The soluble production of protein both in vitro and in vivo has been shown to be directly effected by the ΔF508 mutation, suggesting that the physical properties of the globular NBD1 domain are altered ( 6 , 26 , 30 ). In contrast, analyses of the soluble, native protein have demonstrated that the wild type and ΔF508 NBD proteins are similar with respect to their native state structures. This suggests that the primary effect of the mutation is not a dramatic alteration of native state structure ( 6 , 26 , 30 ).

To determine how the ΔF508 mutation interferes with CFTR folding and structure, we have probed NBD1 production in isolation and in the context of full-length CFTR. These results demonstrate that the biochemical properties of NBD1 are altered in the absence of other CFTR domains by the introduction of the ΔF508 mutation. Rescue of ΔF508 CFTR correlates well with folding and solubility measurements of NBD1 in isolation. In addition, the ability of a novel second-site suppressor, located within TMD2, to rescue the ΔF508 and F508K mutants demonstrates that proper domain-domain assembly is critical to CFTR maturation. Together, these studies suggest that the ΔF508 mutation perturbs multiple steps critical for CFTR maturation.

EXPERIMENTAL PROCEDURES

Full-length cftr expression and pulse-chase analysis.

pCMV-CFTR-Not6.2 expression plasmids, a generous gift from J. Rommens, were mutagenized using the QuikChange site-directed mutagenesis kit (Stratagene) and confirmed by automated DNA sequencing. Expression plasmids were transiently transfected using the FuGENE6 reagent (Roche Applied Science) and allowed to express for 48 h. Twenty-four hours post-transfection, the cells were treated with 5 m m sodium-butyrate. Forty-eight hours post-transfection, the cells were washed once in PBS and lysed in radioimmune precipitation assay buffer (20 m m Tris, 150 m m NaCl, 0.5% w/v deoxycholate, 1.0% v/v IGEPAL CA-630, 0.1% w/v SDS, Complete protease inhibitors (Roche Applied Science), pH 7.9) on ice. The lysates were cleared by centrifugation, analyzed by SDS-PAGE using Tris-glycine gels, and transferred to nitrocellulose for Western blotting. CFTR proteins were probed with L12B4, an α-NBD1 monoclonal antibody, or M3A7, an α-NBD2 monoclonal antibody (Upstate Biotechnology). Blots were developed using Pierce SuperSignal Durawest. Data shown are representative of at least four experiments with each CFTR construct.

Electrophysiological Measurement of Full-length CFTR

The pCMV-CFTR-Not6.2 constructs were transiently expressed in HEK 293T cells that were cultured at 37 °C until experimentation. Excised, inside-out macropatch recordings were performed in the presence of PKA (110units/ml) and ATP (1.5 m m ) at room temperature. All patches were obtained using similar pipette sizes (ca 2 megaohms tip resistances). Recordings were made in symmetric solutions containing 140 m m N -methyl- d -glucamine chloride, 3 m m MgCl 2 , 1 m m EGTA, and 10 m m TES, pH 7.3. Glibenclamide (400 μ m ) was added at the end of each recording to block CFTR channels.

Computational Analysis

CFTR NBD1 was used as a BLAST seed to identify ABC transporter NBD sequences. Approximately 19,000 sequences were used to generate multiple sequence alignments with Clustal consisting of 500 sequences per alignment. The amino acid distribution at specific positions was then assessed in the alignments for both the eukaryota and prokaryota subsets.

Bacterial Yield Assay

Bacterial expression plasmids containing His 6 -Smt3 tagged murine and human NBD1 proteins, containing CFTR residues 389–673, previously described, were used for the in vivo yield assays ( 5 , 26 , 31 ). Overnight donor cultures were diluted 1:50 into LB with 50 mg/ml kanamycin and grown to an OD 600 of 1.0 AU. The cultures were shifted to 25 °C, induced with 750 μ m isopropyl 1-thio-β- d -galactopyranoside, and allowed to grow for 18–20 h. The final OD 600 of each culture was determined, and lysis volumes were adjusted for differences in growth. From the 1-liter cultures, 500 ml were removed, and cells were harvested by centrifugation at 4000 × g relative centrifical force for 15 min at 4 °C. The cells were resuspended in lysis buffer (100 m m Tris, 150 m m NaCl, 5 m m MgCl 2 , 2 m m ATP, 1 m m DTT, 12.5% w/v glycerol, pH 7.6) and sonicated on ice 3 × 1-h intervals with at least 1 h between cycles per sample (Branson Sonifer, 50% duty cycle, output level 5). After sonication, a sample of the lysate was removed for use as the whole cell lysate expression control. The remainder of the lysate was spun for 40 min at 40,000 × g relative centrifical force at 4 °C, and a sample of the supernatant was removed and used as the soluble fraction. Samples were separated by SDS-PAGE on 10% Tris-Tricine gels, transferred to nitrocellulose, and probed with either α-His (Novagen) or α-NBD1 (L12B4, Upstate Biotechnology) monoclonal antibodies and HRP-conjugated α-mouse secondary. Blots were developed with ECL-Plus chemiluminescent reagent and imaged on a GE Healthcare Storm PhosphorImager. The data shown are representative of 4–6 experiments with each NBD construct. Experiments were performed in a single blind manner to exclude contributions of processing order and/or subtle changes in handling.

Mammalian Complementation Assay

The His-Smt3-NBD1 sequence was cloned into a pcDNA vector containing an in-frame fusion of the HA-α sequence. HEK 293 cells were transfected with 1.5 μg of NBD-α and 1 μg of ω-plasmids using FuGENE 6 following the manufacturer's protocols (Roche Applied Science). 24 h post-transfection, cells were treated with 5 m m sodium butyrate. 48 h post-transfection, the cells were washed twice in PBS and lysed by sonication on ice for 15 s (Branson Sonifer, 30% duty cycle, output level 3) in 1× Reporter Lysis Buffer (Promega). The lysates were mixed 1:1 with 1× Reporter Lysis Buffer supplemented with fluorescein-di-β-galactopyranoside (2.5 μ m final concentration), aliquoted into 96-well plates, and read on a SpectraMax Gemini EM at room temperature in kinetic mode.

Limited Proteolysis of CFTR

Microsomes were prepared by nitrogen cavitation and differential centrifugation as described ( 32 ). The final preparations were resuspended in 10 m m HEPES, 0.25 m sucrose, pH 7.5. Microsomes (1–1.5 mg/ml protein) were treated in PBS buffer with the indicated concentrations of trypsin or chymotrypsin for 15 min on ice. The reaction was terminated with 2 m m MgCl 2 , 1 m m PMSF, 5 μg/ml leupeptin and pepstatin. 0.4 mg/ml soybean trypsin inhibitor was added for trypsin digestion. Digested microsomes were analyzed by SDS-PAGE and immunoblotted with the indicated anti-CFTR antibodies. Data shown are representative of at least three experiments for each construct.

Trafficking and Functional Rescue of CFTR by Second-site Suppressor Mutations

The introduction of a mutation or group of mutations within a multidomain protein may have multiple effects on the processing and activity of the complex of domains in the native state. Such is the case with CFTR; the deletion of Phe-508 results in ER retention and subsequent degradation of a biosynthetic intermediate. To ascertain how the ΔF508 mutation alters the conformational maturation of the nascent polypeptide, we probed the folding of CFTR utilizing a series of mutations that are predicted to either effect the folding of NBD1 and/or its association with other CFTR domains to better map the biosynthetic steps that are directly perturbed by the ΔF508 mutation.

The introduction of the single mutations, G550E, R553M or R553Q, and R555K, has previously been shown to partially rescue the ΔF508 trafficking defect in CFTR and restore channel activity at the plasma membrane ( Fig. 1 A ) ( 19 , – 21 ). To evaluate the combined effects of the second-site suppressors with respect to the ΔF508 mutation and identify the mechanism(s) by which they correct the ΔF508 defect, CFTR harboring the various mutations was expressed in transiently transfected HEK-293 cells and analyzed by Western blotting and pulse-chase analyses ( Fig. 1 , B and C ). Maturation of the CFTR polypeptide includes co-translational core glycosylation (band B) in the ER followed by the posttranslational folding/assembly and subsequent complex glycosylation in the Golgi (band C). At steady state, wild type CFTR shows a mixture of both core and complexly glycosylated protein in these heterologous expression systems, indicative of protein at various stages of biosynthesis and trafficking out of the ER and Golgi compartments. The ΔF508 protein appears as only the core glycosylated, band B form, consistent with its retention in the ER. Pulse-chase analyses show the transition from band B to band C in the wild type, wild type -3M, and ΔF508-3M proteins. The ΔF508 protein fails to produce detectable levels of band C CFTR under these conditions. The formation of band C by the wild type, and the -3M-containing variants, is indicative of proper folding, ER-exit, and trafficking of CFTR to the Golgi ( Fig. 1 , B and C).

Rescue of ΔF508 CFTR by -3M mutations. The introduction of the -3M mutations (G550E, R553M, R555K) rescues the trafficking defects associated with the ΔF508 mutation and restores near wild type function. A , a schematic of CFTR showing the five distinct domains and the relative locations of the ΔF508 and suppressor mutations is shown. Transmembrane domains are colored blue , and the nucleotide binding domains are yellow . The locations and residue numbers approximating N- and C-terminal domain boundaries are shown for reference. The location of the Phe-508 position is shown as a red circle , and the four R X R motifs are shown as orange squares . The sequence of the -3M combination of suppressor mutations is shown. B , Western blots of CFTR, expressed transiently in HEK-293 cells, show the maturation defects associated with mutation at position 508 and the rescue of these mutants by the inclusion of the -3M mutations. Band B , core glycosylated protein; Band C , complexly glycosylated protein. C , pulse-chase analysis of the CFTR constructs shows an increase in the production of Band C ΔF508 CFTR in the presence of the -3M suppressors. Wild type and ΔF508 CFTR, both with and without the -3M suppressors, are shown. D , functional rescue of the ΔF508 protein accompanies the rescue of CFTR ΔF508 trafficking as measured in whole cell and macropatch techniques of HEK-293T cells expressing the pCMV-CFTR constructs utilized in B. Measurements were made in the presence of PKA and ATP. Holding potential = −80 mV. Data shown are representative of at least three experiments.

The side chain of the Phe-508 lies at a predicted domain-domain interface between the NBDs and the TMD(s), and maturation of the full-length protein is sensitive to substitutions at this position ( 12 , 17 ). To determine whether the suppressor mutations rescue substitutions at the Phe-508 position, which disrupts CFTR folding without measurable impact on NBD1 folding, the -3M suppressor mutations were introduced into Phe-508 missense proteins ( Fig. 1 B ). The inclusion of the -3M mutations failed to significantly rescue the folding of the F508D and F508K mutants, suggesting that the -3M suppressors do not directly influence the interaction between NBD1 and other domains of CFTR ( Fig. 1 B ). Consistent with this result, the introduction of the -3M mutations onto F508A and F508C had little effect on protein maturation. Interestingly, the -3M mutations rescued the folding and maturation of the F508P protein, which has previously been reported to be refractory to low temperature rescue ( 12 ).

To ascertain the functionality of the -3M-rescued ΔF508 protein, HEK-293T cells were transiently transfected with the pCMV-CFTR constructs, and Cl − currents were measured in excised macropatches. A summary of the functional activity of the WT, ΔF508, and -3M constructs is shown in Fig. 1 D . The introduction of the -3M mutations rescued the function of the ΔF508 protein to near wild type levels, consistent with the trafficking rescue shown in Fig. 1 , B and C . Consistent with prior studies on individual mutations at the 550 and 555 positions, the wild type protein showed an increase in activity when the -3M mutations were introduced ( 20 , 22 ). ΔF508 channel activity for cells cultured at low temperature is shown for reference.

Role of the RXR Motifs within NBD1

Both the ΔF508 and the second-site suppressor mutations are located in NBD1, although the suppressors are distal to the Phe-508 position ( Fig. 2 A ). Previous studies have demonstrated that mutation of the R X R motifs in CFTR improves the trafficking and maturation of ΔF508 CFTR, although the mechanisms behind this rescue are not fully understood ( 22 , 33 ).

Role of R555 in CFTR maturation. A , the -3M second site suppressor mutations are located within the NBD1 domain distal to the Phe-508 locus and do not directly contribute to the surface or structure altered by the deletion of the phenylalanine. Two views of NBD1 are shown rotated ∼90° with respect to one another. The Phe-508 position is shown in red , and the location of the second site suppressor positions are shown in blue. B , analysis of the R X R domains within NBD1 shows high conservation at the 555 position, consistent with its role in the ABC transporter signature motif but much lower conservation at the 516, 518, and 553 positions. C , substitution of the Arg-555 position alters wild type CFTR trafficking. The substitution of R555A, R555G, and R555T resulted in a marked reduction in the formation of band C CFTR, whereas the R555K, as measured by Western blotting of transiently transfected HEK-293 cells displays near wild type CFTR maturation. Data shown are representative of at least four independent experiments.

To evaluate the evolutionary conservation of the R X R sequences in NBD1, an alignment of more than 19,000 NBD sequences was generated, and sequence conservation at the CFTR NBD1 R X R motif sites was then assessed. The alignment is of high quality at the 553 RAR 555 sites as these sequences immediately follow the highly conserved LSGGQ motif. The 553 R X R 555 sequence is also highly conserved and has been attributed to an extended ABC signature motif sequence (LSGGQ XX R). The Arg at the 555 equivalent position is conserved across >75% of the NBD sequences in alignments containing eukaryotic and prokaryotic NBD sequences. The 553-equivalent position is also conserved with respect to three amino acids: arginine, lysine, and glutamine. Interestingly, a second R X R motif, 516 R Y R 518 is not well conserved across these diverse alignments (data not shown).

Previous studies have shown that single-site substitution of the second Arg in an R X R motif facilitates trafficking of other membrane proteins ( 34 , 35 ). Disruption of the R X R by substitution of Arg-555 with lysine showed no discernible effects on wild type CFTR maturation. By contrast, substitution with alanine, glycine, or threonine in wild type CFTR resulted in the significant decrease in band C protein. The loss of a retention/retrieval signal would not be predicted to alter the trafficking of wild type CFTR, which normally traffics in the presence of such a signal.

Effects of the -3M Mutations on NBD1

To ascertain the effect that the ΔF508 and the -3M mutations have on the NBD itself, these mutations were evaluated using NBD1 protein expressed heterologously in Escherichia coli in isolation from other CFTR domains or eukaryotic quality control proteins. Previous studies have described the expression, purification, and crystallization of NBD1 utilizing a Smt3-based fusion system and have qualitatively indicated that the ΔF508 NBD1 protein is either insoluble in these systems or is significantly less soluble than wild type ( 5 , 26 ).

NBD1 was expressed in E. coli , and the quantity of soluble protein was determined by Western blotting after controlling for differences in culture growth and separation by high speed centrifugation. The ΔF508 mutation decreased the soluble production of NBD1 protein ( Fig. 3 A , SOL ), although total expression was unaffected by the ΔF508 mutation (whole cell lysate). Quantification of the soluble fraction of NBD1 shows a three- to 5-fold difference in the soluble quantities of wild type and ΔF508 NBD1, consistent with a direct effect of the ΔF508 mutation on NBD1 ( Fig. 3 B ).

Influence of the ΔF508 and -3M mutations on NBD1 folding. A and B , expression of the NBD1 protein in E. coli is directly affected by the inclusion of the ΔF508 and the -3M mutations. NBD1 protein was expressed as a fusion with an N-terminal His-Smt3 and assayed by Western blotting after sonication and centrifugation. The soluble protein samples ( SOL ) are clarified by centrifugation at 40,000 × g relative centrifical force, and the whole cell lysates ( WCL ) are shown as controls for expression and loading. Representative data are shown. B , soluble production of NBD1 in HEK-293 cells is influenced by the introduction of the ΔF508 and -3M mutations, as measured by β-galactosidase enzymatic activity. Changes in signal intensity reflect changes in soluble NBD production and enzymatic activity. Quantification of the end point β-galactosidase data is presented. Data shown are the mean and S.D. from at least 12 experiments for each mutant.

The -3M mutations were then introduced into both the wild type and ΔF508 NBD1 backgrounds. The introduction of these mutations caused a significant increase in the quantity of soluble protein ( Fig. 3 A , SOL ); again, total expression levels were unchanged with the inclusion of the -3M mutations (whole cell lysate). The -3M mutations increased the soluble quantity of NBD1 protein under identical expression conditions in both wild type and ΔF508 NBD1 ( Fig. 3 A ). The magnitude of this effect was similar for both wild type and ΔF508 NBD1 proteins and was similar with both Arg-553 substitutions (Met and Gln). These data demonstrate that the ΔF508 and suppressor mutations alter the properties of the NBD1 protein in the absence of other CFTR domains, and these effects are independent of the mammalian chaperone systems.

To probe the effects of these mutations in a mammalian system, NBD1 was expressed in HEK-293 cells and monitored by a β-galactosidase folding/solubility assay ( 36 ). This enzymatic assay relies on the complementation of the α-fragment of β-galactosidase, fused to the target protein (Smt3-NBD1), with the ω-fragment of β-galactosidase, expressed independently. Enzymatic activity has been shown previously to correlate with the production of soluble protein ( 36 , 37 ). Transient cotransfections of the Smt3-NBD1-α constructs and the ω-fragment of β-galactosidase were performed, and activity was monitored utilizing a fluorogenic substrate, fluorescein-di-β- d -galactopyranoside.

When co-expressed with the ω-fragment, wild type Smt3-NBD1-α fusion proteins produced fluorescence significantly above controls, including HEK293 background and cells expressing only the ω-fragment. The inclusion of the ΔF508 mutation into this construct significantly decreased the relative fluorescence signal, p value <0.005 (Welch's analysis of variance)( Fig. 3 B ). Western blot analysis of the whole cell lysates showed that both proteins were expressed, and levels of soluble expression correlated with relative enzymatic activity, consistent with differential solubility and/or turnover of the wild type- and ΔF508-α fusion proteins (data not shown).

The -3M second-site suppressors were then introduced into the Smt3-NBD1-α constructs and evaluated for their ability to influence soluble protein production in this system. Similar to the results seen in bacteria, the introduction of the second-site suppressor mutations into NBD1 in mammalian cells produced an increase in enzymatic activity for both the wild type and ΔF508 protein fusions ( Fig. 3 B ). The fluorescence signal of the ΔF508–3M-α fusion proteins increased ∼2.5-fold relative to ΔF508 to near wild type levels ( p < 0.005). The -3M mutations also increased the wild type NBD1-α signal 2.3-fold ( p < 0.005). Western blot analyses of the NBD1-α fusion proteins showed a strong correlation between expression levels of the fusion proteins and the corresponding enzymatic activities (data not shown).

Structural Analysis of CFTR

To evaluate the effects that the ΔF508 and -3M mutations have on the structure of full-length CFTR, limited proteolysis of CFTR stably expressed in BHK cells was performed and analyzed using antibodies specific to epitopes in NBD1 (660, L12B4) and NBD2 (M3A7). Previous studies have indicated that the ΔF508 mutation has adverse effects on the structure of the cytoplasmic domains of full-length CFTR ( 12 ). Specifically, the inclusion of the ΔF508 mutation in NBD1 increased the proteolytic sensitivity of NBD2 while only modestly altering the proteolytic cleavage of NBD1, as measured with the M3A7 and L12B4 antibodies, respectively.

Limited digestion of CFTR with chymotrypsin was performed, and the relative proteolytic stabilities of the NBD1 and NBD2 domains were probed with the L12B4 and M3A7 antibodies, respectively. As previously described, the inclusion of the ΔF508 mutation within the NBD1 sequence dramatically altered the production of a stable NBD2 fragment containing the M3A7 epitope ( Fig. 4 A ). The M3A7 epitope-containing NBD2 fragment (∼30 kDa) decreased dramatically in the ΔF508 protein but was partially restored when the -3M mutations were included in the ΔF508 background ( Fig. 4 A , M3A7 ). Similarly, in analysis with the L12B4 (NBD1) antibody, a cluster of bands at ∼35 kDa showed sensitivity to the ΔF508 mutation that was restored by the -3M mutations ( Fig. 4 A , L12B4 ).

Structural analysis of CFTR wild type, ΔF508, and suppressed proteins. Limited proteolysis utilizing either trypsin or chymotrypsin was performed to assess the stability of the cytosolic NBD domains. A , a chymotrypsin digestion of CFTR expressed in BHK cells was probed with either L12B4 (residues 385–410) or M3A7 (residues 1373–1382). B , a trypsin digest of CFTR expressed in BHK cells is shown probed with the NBD1 L12B4 and 660 antibodies. The proteolytically stable, putative NBD1 bands are highlighted within dashed lines .

Recent studies have suggested that the L12B4 epitope may report on the proteolytic susceptibility of both NBD1 and TMD1 ( 38 ). The L12B4 epitope (residues 385–410) is located in the extreme N terminus of NBD1 and is separated from the core of NBD1 by a large disordered loop (residues 410–430) that is likely susceptible to early cleavage. Thus, to further assess the proteolytic sensitivity of NBD1 in full-length CFTR, limited trypsinolysis was performed, and blots were probed with both L12B4 and a second antibody, Ab660, whose epitope lies between residues 484–589 in the core of NBD1 ( Fig. 4 B ).

Analysis with the 660 antibody clearly demonstrated the formation of a stable, NBD1 core epitope-containing band of ∼30 kDa ( Fig. 4 B , boxed ). The degradation of this product was significantly increased by the ΔF508 mutation and stabilized by the inclusion of the -3M mutations in the ΔF508 background. Consistent with the chymotrypsin results and previous studies, analysis of the trypsin digestion with the L12B4 antibody yields a predominant band of ∼40 kDa in the wild type, ΔF508, and -3M proteins ( Fig. 4 B , boxed ). The intensity of this band was decreased, and the band appeared to be more susceptible to proteolytic degradation in the ΔF508 as a function of the total amount of CFTR present in the undigested lane. Furthermore, a band of ∼20 kDa appeared in the ΔF508-containing samples and was decreased with the inclusion of the -3M suppressor mutations. The Ab660 antibody failed to identify the ∼40-kDa products associated with the L12B4 antibody, suggesting that the core sequence of the NBD was not included in this digested fragment.

NBD-NBD Interactions Involved in CFTR Maturation

The proteolytic data indicate that NBD2 conformation is sensitive to the ΔF508 mutation in NBD1 and that the suppression of the ΔF508 defects by the -3M mutations partially restores the native, proteolytically resistant conformation to NBD2. It is not known how this occurs nor is it known what role NBD2 plays in the recognition of the ΔF508 mutation by the quality control machinery. To assess the potential role that NBD dimerization plays in CFTR maturation, mutations within the ATP-binding sites were introduced into pCMV-CFTR plasmid and expressed in HEK293 cells.

ABC-transporter NBDs have been shown to dimerize during their ATP binding and hydrolysis cycles ( 39 , 40 ). The canonical ATP binding sequences associated with ABC transporters include the Walker A and B sequences and the signature motif ( Fig. 5 A ). Mutations of the Walker A lysine (K464A and K1250A in NBD1 and NBD2, respectively) have been shown to dramatically decrease ATP affinity ( 40 ). Conversely, mutation of the catalytic glutamate to glutamine in NBD2, E1371Q, has previously been shown to stabilize NBD dimers by trapping ATP at the NBD-NBD interface ( 39 ). Both sets of mutations were evaluated in the wild type and ΔF508 backgrounds after transient transfection and expression in HEK 293 cells to assess the potential role(s) of NBD heterodimerization in CFTR trafficking ( Fig. 5 , A and B ).

NBD-NBD interactions in CFTR maturation. A , a schematic of ATP-binding sites and the associated binding and dimerization events determined in NBD proteins are shown. Composite sites (Walker A and B) and the ABC transporter signature motif are labeled A , B , and C , respectively. B , mutation of the composite ATP-binding site in NBD1, K464A, adversely affects the trafficking of wild type CFTR. Mutation of the equivalent position in NBD2, K1250A, has minimal effect on CFTR maturation. The NBD-dimer stabilizing mutation, E1371Q, does not dramatically alter the trafficking of the wild type or ΔF508 CFTR proteins when expressed transiently in HEK-293 cells. C , deletion of NBD2 does not dramatically alter the trafficking efficiency of wild type relative to ΔF508 either with or without the -3M mutations. Core ( Band B ) and complexly glycosylated protein ( Band C ) are indicated by B * and C * to reflect changes in molecular weight. Data shown are representative of four experiments.

Mutations made in the NBD2 composite ATP-binding site, the functionally active ATP site, had no dramatic effect on CFTR trafficking ( Fig. 5 B ). Stabilization of the putative NBD1-NBD2 dimer via the E1371Q mutation did not facilitate the trafficking of the ΔF508 protein and had no discernible effect on the maturation of the wild type protein. Similarly, the introduction of the K1250A mutation had minimal effects on the maturation of wild type CFTR and failed to rescue the ΔF508 CFTR protein. The NBD1 K464A mutation also failed to rescue ΔF508 trafficking. However, when introduced into the wild type background, the K464A reduced CFTR maturation, as evidenced by a decrease in band C.

Although stabilization of the NBD1-NBD2 interaction had little effect on CFTR trafficking, it was possible that NBD2 plays a key role in the suppression of the ΔF508 defects by the -3M mutations ( 12 ). To test this hypothesis, a ΔNBD2 CFTR protein previously employed to demonstrate that NBD2 is not required for the maturation of full-length wild type CFTR ( 41 ), was utilized. Other studies have demonstrated that maturation of similar ΔNBD2 CFTR proteins are sensitive to the inclusion of the ΔF508 mutation, although the deletion of NBD2 interferes with low temperature rescue ( 38 , 42 , 43 ).

Consistent with those findings, the deletion of NBD2 did not inhibit the trafficking of wild type CFTR, as evidenced by the presence of band C ΔNBD2 CFTR ( Fig. 5 C ), and the ΔF508 mutation caused the loss of band C, consistent with its retention in the ER. The -3M mutations promoted the folding and trafficking of the ΔF508-ΔNBD2 CFTR protein, as evidenced by the presence of band C by Western blotting, indicating that their effect does not require NBD2.

TMD-NBD Interactions in CFTR Maturation

Recent studies have suggested that the core CFTR structure, capable of exiting the ER, is formed by the protein sequences including the N terminus, TMD1, NBD1, R, and TMD2 but does not require NBD2 ( 38 , 41 , – 43 ). To evaluate the possibility the interdomain interactions in the core CFTR structure could be effected by ΔF508, models of the NBD-TMD interactions in CFTR were produced using the extant Sav1866 crystal structures. These CFTR structural models indicate that the first and fourth intracellular loops (ICL1 and ICL4) are predicted to interact with NBD1, with ICL4 in close proximity to the Phe-508 residue side chain. Based on sequence alignments and homology modeling, sites within ICL4 were chosen to probe the NBD-TMD interactions in wild type and mutant CFTR ( Fig. 6 A ). A single tryptophan residue was introduced at positions within ICL4 and evaluated for its ability to rescue the ΔF508 trafficking defect ( Fig. 6 B ). A tryptophan residue was chosen with the hypothesis that the aromatic side chain would physically fill the void created by the ΔF508 mutation, increasing the affinity of the TMD-NBD interaction. The substitution of R1070W had little effect on the maturation of wild type CFTR but measurably promoted trafficking of ΔF508 CFTR ( Fig. 6 B ). Substitution of either Lys or Ala at the 1070 position did not facilitate maturation of ΔF508 CFTR.

NBD-TMD interactions in CFTR maturation. Mutations in ICL4 at position 1070 were evaluated for effects on the trafficking of wild type, ΔF508, and F508K CFTR, transiently expressed in HEK293 cells. As previously described, the presence of the higher molecular weight band, Band C, is indicative of proper folding and trafficking to the Golgi. A , a model of ICL4-NBD1 interactions was derived from sequence alignments and the Sav1866 crystal structure (2HYD). NBD is shown in green , and ICL4 is shown in blue . The Phe-508 and Arg-1070 residue side chains are shown in red . Two views, rotated by 90 degrees are shown. B , Western blots show the effects of the ICL4 Arg-1070 mutations on the trafficking of wild type, ΔF508, and F508K CFTR. C , the R1070W and -3M suppressor mutations were evaluated for their ability to rescue the ΔF508 mutation either independently or in combination. The inclusion of the -3M and R1070W mutations in combination rescued more ΔF508 CFTR than either suppressor set alone. Cells were cultured at 37 °C and evaluated by Western blotting using the M3A7 antibody. D , trafficking of the F508K missense protein was evaluated with the R1070W mutation. Trafficking of F508K was partially rescued by the R1070W mutation. Data shown are representative of at least four experiments.

To evaluate the potential mechanisms by which the R1070W mutation rescued ΔF508, this mutation was also introduced into the ΔF508–3M and F508K backgrounds ( Fig. 6 , C and D ). F508K is expected to disrupt the interdomain interaction, as it interferes with maturation but does not affect the isolated NBD1 ( 26 ). The combination of the -3M mutations with the R1070W mutation increased ΔF508 Band C production, as compared with ΔF508–3M and ΔF508/R1070W alone. The increases in Band C production suggest distinct or independent mechanisms of action. In this regard, the R1070W mutation induced the formation of Band C in the F508K mutant predicted to disrupt the interdomain interaction, an effect not seen for low temperature or with the -3M mutations ( Fig. 6 D ).

CFTR biosynthesis is a complex process that requires the folding and subsequent assembly of multiple independent domains. Utilizing a series of experiments aimed at identifying the defects associated with the ΔF508 mutation and its suppression by second-site mutations, a more detailed model of the folding pathway of CFTR has been generated ( Fig. 7 ). In this model the primary manifestation of the ΔF508 defect lies within the NBD1 domain itself, which has previously been shown to fold co-translationally and autonomously of other CFTR domains. In the model, failure of this domain to fold and then stably associate with the core CFTR structure (TMD1-NBD1-R-TMD2) results in altered CFTR-chaperone interactions, ER retention, and ultimately, degradation of CFTR.

A model for CFTR maturation and the influence of suppressor mutations in NBD1 and TMD2. The association of NBD1 with both CFTR TMD components and cellular quality control is in dynamic equilibrium. Decreases in NBD solubility as a result of inefficient folding due to the ΔF508 mutation results in an increase in QC-NBD association. The prolonged NBD-QC interaction ultimately leads to ERAD of CFTR; lower pathway. The -3M suppressor mutations decrease the QC-NBD interaction by stabilizing and/or solubilizing NBD1. The R1070W mutant in TMD2 suppresses ΔF508 by promoting the interactions between NBD1 and ICL4 as required for maturation. By relieving the QC-NBD interactions, the -3M and R1070W mutations promote CFTR maturation; upper pathway . The ER export- competent CFTR structures, full-length, and ΔNBD2 are shown boxed in the upper right .

A series of mutations were introduced into NBD1 and full-length CFTR to probe the role of NBD1 folding and interdomain association in CFTR maturation. Second site suppressors improve folding of the isolated NBD1 and were used to assess the role of domain folding in cellular models, including E. coli and HEK293 mammalian cells. To complement these, novel suppressing mutations in the fourth intracellular loop (in TMD2) were used to assess the requisite domain-domain interactions needed for CFTR maturation when expressed in HEK293 cells.

The introduction of the -3M second site suppressors into full-length CFTR provides several key insights into the CFTR biosynthetic pathway. First, the mechanism(s) underlying the defects associated with the missense and ΔF508 mutations is different. The suppression of the ΔF508 and F508P substitutions, but not the F508D and F508K mutants, indicates that these mutations alter CFTR folding by discrete mechanisms or are of differing severities. This is consistent with the structural models based on bacterial ABC transporters wherein the Phe-508 residue packs against an ICL from TMD2 (ICL4). The loss of Phe-508 is predicted to alter the geometry of the surface without significant alteration to its hydrophobicity. Alterations in chemical character within this interface are predicted to alter this association. Consistent with this, structures of NBD1 F508S and F508R and trafficking of F508S and F508R full-length CFTR demonstrate that the severity of physicochemical alterations at the 508 position correlate with protein trafficking ( 12 , 26 ). These data suggest that this putative domain-domain interface is critical to the maturation of the CFTR protein.

Second, the loss of the Phe-508 side chain can be accommodated by full-length CFTR when complemented by the NBD1 suppressor mutations. The rescue of ΔF508 NBD1 and CFTR without the full restoration of the NBD-ICL interface ( i.e. physical replacement of the missing Phe side chain) suggests that NBD properties are altered more generally. Restoring these physical properties ( i.e. solubility, dynamics, stability), NBD-TMD interactions facilitates maturation even though the surface of NBD1 is not.

Finally, rescue of the ΔF508 defect can also be accomplished via alterations within multiple CFTR domains. Domain-domain association events are critical to forming the core CFTR structure. Facilitating these interactions from either side of the interaction interface putatively restores the requisite domain-domain association, thereby promoting CFTR maturation. Furthermore, either improving the folding of one or both of the domains forming the surface or stabilizing the interaction itself would be reflected as increased maturation.

The refined model of CFTR folding and maturation highlights these individual events ( Fig. 7 ). NBD1 is capable of associating with the core structure of CFTR when folded and with cellular chaperone systems when misfolded or partially folded in dynamic equilibria. The inclusion of the ΔF508 mutation alters these equilibria, resulting in prolonged NBD1-chaperone interactions. Failure of the NBD to associate with the core structure of CFTR may also be recognized by cellular quality control systems as alterations in TMD structure. NBD2 seems to be particularly sensitive to a disruption of these interactions. The loss of the appropriate folding and association results in the lower branch of the folding model wherein NBD1 fails to complement other domains in the core CFTR structure. These events ultimately lead to ERAD targeting of CFTR to the proteasome.

Rescue of ΔF508 may be accomplished by facilitating NBD1 folding directly or by stabilizing its interactions with the core CFTR structure. The -3M suppressor mutations directly alter the biochemical properties of the NBD in isolation ( Fig. 3 ). The increased solubility and reported stability potentially shift the equilibria toward a native NBD state, capable of interacting with the core CFTR structure. This association is putatively critical to completing the CFTR core structure ( Fig. 7 , upper arm ). In the model, by stabilizing and/or improving the yield of the native NBD fold, the -3M mutations also facilitate appropriate domain-domain interactions. This may occur indirectly via decreasing NBD1-chaperone interactions, thereby allowing a physical interaction between NBD1 and the TMDs. More likely, the -3M suppressors act directly via stabilizing the native NBD fold, thereby increasing its availability and indirectly promoting domain-domain interactions.

To further probe the role of the -3M mutations in NBD1, a bioinformatic analysis was used to evaluate the Arg-553 and Arg-555 positions across a large alignment of sequences. Analysis of the R X R sequences suggests an evolutionarily conserved role for the di-arginine residues at these positions. It is possible that these residues were also utilized to serve as retention/retrieval sequences in eukaryotes, as has been previously suggested. However, bioinformatic analyses demonstrate conservation across both prokaryotic and eukaryotic sequences, consistent with a basic structural role. Specifically, the conservation at the Arg-555 position (>75% in both eukaryotes and prokaryotes) suggests strong evolutionary pressure for Arg at this site. As the conservation at this site is preserved in both eukaryote and prokaryotes, the evolutionary pressure exerted on this position is independent of cellular trafficking machinery.

The decrease in wild type CFTR trafficking seen with the R555A/G/T demonstrates that the basic side chain at the 555 locus is required for proper trafficking. The loss of CFTR trafficking with the R555A/G/T mutations suggests this site defines more than a simple signal motif. Consistent with these data, the R555G mutation has previously been identified in a heterozygous CF patient (R555G/Y1307X). Furthermore, structural and functional roles of the Arg-555 residue have been identified in a homologous system wherein intradomain movements are coordinated by interactions between the Arg side chain and a neighboring Asp residue. Together, these data suggest that the conserved Arg-555 residue plays a critical role in NBD structure and function.

Proteolytic analyses also demonstrate that the -3M mutations alter the structure of the wild type and ΔF508 CFTR proteins ( Fig. 4 , A and B ). Changes in NBD1 proteolysis, as a result of the ΔF508 mutant are partially reverted by the -3M mutations, as evidenced by stabilization of both NBD1 and NBD2 bands in trypsin and chymotrypsin digests. Furthermore, these changes in proteolytic protection and CFTR maturation correlate with the changes in NBD1 solubility ( Figs. 2 , ​ ,3, 3 , and ​ and4), 4 ), providing a structural correlate for the alterations in solubility and stability within NBD1. Reversion of the NBD2 proteolytic sensitivity suggests that appropriate domain-domain interactions (and global structure) are partially restored by the -3M mutants. This is consistent with a model wherein proper NBD1 folding in turn facilitates appropriate domain-domain interactions and global CFTR structure.

To evaluate the possibility that domain-domain association and stabilization could be facilitated by stabilization of the NBD1-NBD2 heterodimer, mutations that are known to alter NBD oligomerization and channel gating were introduced into wild type and ΔF508 CFTR ( Fig. 5 A ). Stabilization of the NBD heterodimer by the E1371Q mutation had no discernible effect on wild type or ΔF508 maturation ( Fig. 5 B ). The failure of the E1371Q mutant to rescue ΔF508 CFTR is consistent with ΔF508 influence on the early step of NBD1 folding and that stabilization of the NBD-NBD dimer may not facilitate ΔF508 maturation.

As well, disruption of the composite ATP-binding site in NBD2 by the K1250A mutant had no discernible effect on CFTR maturation. In contrast, the K464A NBD1 ATP-binding mutant decreased wild type CFTR maturation. These data are consistent with previous reports of effects of Lys-464 mutants on maturation and support a model wherein ATP serves as a structural co-factor for NBD1 but not NBD2.

Analysis of ΔNBD2 CFTR suggests that CFTR trafficking is generally refractory to the presence of NBD2. Both the ΔF508 and -3M mutants behaved similarly when expressed in full-length or ΔNBD2 CFTR. The ΔF508 ΔNBD2 protein failed to produce significant quantities of band C but was partially rescued when the -3M mutations were introduced. However, the observation of less band C in ΔF508 ΔNBD2–3M protein relative to ΔF508–3M suggests that the NBD2 has minimal effects on CFTR maturation and/or stability, although it is itself profoundly impacted by the folding status of the rest of the molecule.

To evaluate the possibility that ΔF508 CFTR could be rescued by stabilization of other domain-domain interactions within the minimal core structure of CFTR, structural models were produced to identify positions critical to the NBD1-TMD interface. Sites within ICL4 were chosen based on their proximity to the Phe-508 residue. The identification of a single site, Arg-1070, within ICL4 that promotes ΔF508 maturation suggests that the NBD1-TMD2 interaction is critical for CFTR biosynthesis.

Although both the Arg-1070 and -3M suppressors rescue ΔF508, suppression by the R1070W mutation is likely accomplished by independent mechanisms. The properties of NBD1 are not directly altered by the Arg-1070 locus. Thus, the R1070W mutation putatively promotes appropriate domain-domain associations by increasing hydrophobic interactions (affinity) at the NBD1-ICL4 domain-domain interaction surface. The relatively hydrophobic surface proximal to the Phe-508 position could potentially accommodate the hydrophobicity and volume of the R1070W substitution. Decreases in side-chain volume (Ala/Gly/Thr substitution) and the presence of charge (Arg-1070 and R1070K) fail to facilitate ΔF508 trafficking. Maturation of the F508K CFTR molecule was potentially facilitated by interactions between the indole side chain from R1070W and the NBD1 surface. An increase in domain-domain affinity as a result of increased hydrophobic interactions may overcome the repulsive forces introduced by the Lys side chain.

Intriguingly, the R1070W mutation, which rescues ΔF508 CFTR, has been identified in patients with mild disease (congenital bilateral absence of the vas deferens, pancreatic sufficient CF). Previous studies suggest that in the wild type background the R1070W mutation alters protein expression, localization, and function ( 44 ). It is possible that changes in interdomain dynamics contribute to ΔF508 maturation and altered wild type CFTR properties, although further study is needed to fully evaluate this possibility.

Taken together, these data suggest that the formation of a core CFTR molecule, including TMD1-NBD1-R-TMD2, is critical for CFTR maturation. The assembly of this core structure requires the proper folding of individual domains and the proper assembly of these domains. Alterations to the processes of domain folding and assembly may both contribute to the misfolding and rescue of ΔF508 CFTR. These data provide evidence for the direct biochemical and biophysical alterations of NBD1 with the ΔF508 and -3M suppressor mutations, demonstrating a critical role of NBD1 folding in CFTR maturation. Modulation of NBD1 folding, therefore, represents an attractive therapeutic target for ΔF508 CFTR. Additionally, the identification of a novel second site suppressor within TMD2 provides evidence that stabilization of the global fold may facilitate NBD1 folding via coupled folding and assembly events, providing additional regions of CFTR that could be targeted for the rescue of ΔF508 CFTR folding and biosynthesis.

Acknowledgments

We thank David Gadsby for suggesting the use of the NBD dimer stabilizing E1371Q mutation and members of the Thomas laboratory for critical comments and helpful suggestions.

* This work was supported, in whole or in part, by National Institutes of Health NIDDK Grants 49835 (to P. J. T.) and 75302 (to G. L. L.). This work was also supported by the Cystic Fibrosis Foundation (to P. J. T.) and the Canadian Cystic Fibrosis Foundation (to G. L. L.). The authors declare they have competing financial interests. The β-galactosidase assay has been licensed to Reata Pharmaceuticals by The University of Texas Southwestern Medical Center at Dallas. P. J. T. is a founding scientist of Reata Pharmaceuticals.

5 The abbreviations used are: CFTR cystic fibrosis (CF) transmembrane conductance regulator ER endoplasmic reticulum ERAD ER-associated degradation QC quality control ICL intracellular loop NBD nucleotide binding domain TES 2-{[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]amino}ethanesulfonic acid TMD transmembrane domain.

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• Published: 15 November 2021

CyFi-MAP: an interactive pathway-based resource for cystic fibrosis

• Catarina Pereira 1 , 2 ,
• Alexander Mazein 3 , 4 ,
• Carlos M. Farinha 1 ,
• Michael A. Gray 5 ,
• Karl Kunzelmann 6 ,
• Marek Ostaszewski 3 ,
• Irina Balaur 3 , 4 ,
• Margarida D. Amaral 1 &
• Andre O. Falcao 1 , 2  

Scientific Reports volume  11 , Article number:  22223 ( 2021 ) Cite this article

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• Cellular signalling networks
• Chloride channels
• Mechanisms of disease
• Molecular medicine
• Systems biology

Cystic fibrosis (CF) is a life-threatening autosomal recessive disease caused by more than 2100 mutations in the CF transmembrane conductance regulator (CFTR) gene, generating variability in disease severity among individuals with CF sharing the same CFTR genotype. Systems biology can assist in the collection and visualization of CF data to extract additional biological significance and find novel therapeutic targets. Here, we present the CyFi-MAP—a disease map repository of CFTR molecular mechanisms and pathways involved in CF. Specifically, we represented the wild-type (wt-CFTR) and the F508del associated processes (F508del-CFTR) in separate submaps, with pathways related to protein biosynthesis, endoplasmic reticulum retention, export, activation/inactivation of channel function, and recycling/degradation after endocytosis. CyFi-MAP is an open-access resource with specific, curated and continuously updated information on CFTR-related pathways available online at https://cysticfibrosismap.github.io/ . This tool was developed as a reference CF pathway data repository to be continuously updated and used worldwide in CF research.

Introduction

Omics technologies revolutionized the way researchers generate data 1 . The integration of disease-specific data allows not only to capture knowledge at its multiple levels of organization but also to unbiasedly identify molecular features, such as phenotypes associated with complex and heterogeneous diseases—which would remain otherwise unnoticed 2 , 3 . The visual representation of molecular mechanisms has emerged as a powerful tool for a better understanding and analysis of specific disease-causing features. This led to the development of several pathway-based resources, such as KEGG (Kyoto Encyclopedia of Genes and Genomes) 4 , Wikipathways 5 , Reactome 6 and MetaCore™ 7 , 8 , 9 , 10 .

The need for dedicated knowledge maps as tools for the representation of mechanisms involved in specific diseases in a holistic form gave origin to the concept of a disease map. A disease map consists of a representation of disease mechanisms illustrating the major signalling, metabolic and regulatory pathways known to be involved in a specific disorder in an interchangeable format. The major feature of the disease map is to allow comparisons among different maps using a high-quality representation in a standardised format 11 . Furthermore, it is computer-readable, user-friendly and can be transformed into mathematical models for predictive analysis and new hypothesis generation 11 . This resource can store and display multiple layers of information—from subcellular, cellular, tissue, organ to whole organism systems—besides providing customizable levels of detail that can be used in the illustration of the biological mechanisms 12 . In the past, to further understand their mechanisms and potentially find therapeutic targets, several disease maps for complex disorders have been developed such as Parkinson’s 13 , Alzheimer’s 14 , asthma 15 , several types/forms of cancer 16 and rheumatoid arthritis 17 .

Cystic Fibrosis (CF) is a life-shorting rare genetic disorder affecting 90,000 to 100,000 individuals worldwide, that results from over 2,100 variants in the CF transmembrane conductance regulator ( CFTR ) gene 18 , 19 , 20 . The CFTR protein functions as a chloride/bicarbonate channel activated through cAMP-induced phosphorylation at the apical plasma membrane (PM) of epithelial cells. This protein works in a dynamic network, interacting with multiple components and regulating a significant number of other channels 21 , 22 , 23 .This channel loss of function causes the organs in which it is expressed to be impacted. Specifically, in the lung CFTR malfunction causes severe airway dehydration and thickening of the lung mucus, leading to impaired mucociliary clearance (MCC) and, subsequently, clogging of the airways. This causes progressive loss of lung function, which is the major cause of morbidity and mortality 24 , 25 . Furthermore, it is important to note that CFTR loss of function does not occur only in CF, but also in chronic obstructive pulmonary disease (COPD)—the third main cause of death worldwide 26 —revealing the crucial role this protein plays in the airway epithelium 27 .

The most common CF-causing mutation on the CFTR gene is the c, which occurs in approximately 70% of people with CF worldwide 28 . F508del- CFTR is a misfolded unstable protein, which is mostly retained in the endoplasmic reticulum (ER) and targeted for ER-associated degradation (ERAD). However, if F508del- CFTR bypasses this pathway and is rescued to the PM, the protein still presents defective channel gating and low PM stability, being rapidly endocytosed and degraded 29 , 30 , 31 . The biogenesis and intracellular trafficking of F508del- CFTR protein has been extensively studied to better understand the pathways that contribute to and drive CF progression. Notwithstanding, and owing to the inherent complexity of the mechanisms and pathways, the CFTR dynamic network is still only partially understood 20 , 32 , 33 , 34 , 35 .

Several databases and software tools have been used in efforts to represent the known CF mechanisms through the representation of pathways covering different processes—such as MetaCore™ from Clarivate Analytics (i.e., CFTR folding and maturation), Reactome (i.e., RHO GTPases regulate CFTR trafficking), KEGG (i.e., ABC transporters—Homo sapiens) and Wikipathways (i.e., ABC-family proteins mediated transport) 6 , 8 , 36 . Notwithstanding, these pathway-based databases present CFTR data incorporated with non-related processes and proteins such as other ABC-family proteins, which although allow comparison between some members of this family, may interfere with the reading and interpretation of CFTR specific data. Furthermore, most are not detailed and/or updated with recent disease features, besides not being freely accessible, nor containing CFTR variants information or allowing comparison between a wt and mutant protein interactions, essential to understanding the progression of this disease. yFi-MAP is developed and publicly available as a complementary resource to the existing tools. Moreover, CyFi-MAP content benefits from being discussed and approved by domain experts (available on the GitHub page, section Team). The incorporation of CFTR molecular mechanisms in a single resource represented in a standardized and inter-exchangeable manner represents one of the main advantages of CyFi-MAP. This tool allows the user to: (i) follow CFTR pathways; (ii) acquire its functional context and subcellular localization through the visualization of the cell compartments represented in the map; (iii) interpret differences between wt-CFTR and F508del- CFTR where unique interactions with CFTR variant are highlighted.

With this goal in mind, in this work, we aimed to build a repository of the available CFTR -related knowledge as a disease map named CyFi-MAP. CyFi-MAP is distinctive from other CFTR databases by being the first CF disease map and aiming to archive molecular mechanisms and biological pathways reported to be relevant to CFTR in a standard and consistent way. This repository is continuously updated with careful manual annotations and is expected to expand in both molecular mechanisms and in CFTR mutations representation and to be continuously updated based on the published literature. Through the map, it is possible to visualize CFTR pathways in protein cycle processes described in a sequential network, resulting in a more global biological interpretation and a faster understanding of CF pathophysiology.

Concept and features

CyFi-MAP offers a resource that accurately (i.e., adequately confirmed in the literature) and graphically illustrates CFTR molecular pathways in an easy-to-read manner by the scientific community. Given that CF is caused exclusively by mutations in the CFTR gene that alter multiple cellular functions, the CyFi-MAP information was organized according to the CFTR life cycle, from its biogenesis to degradation, where two sub-maps were developed: (i) one representative of wt- CFTR and (ii) one of F508del- CFTR .

The main differences between these submaps are given by the representation of some of the key processes side-by-side (available on the website in the Map section), in order to facilitate submap comparison at molecular mechanism level. Additionally, a scheme is also available with CFTR traffic pathways inside the cell where the major physical alterations (e.g., traffic impairment and mucus clogging) in wild-type vs the mutant in airway epithelial cells are depicted (Fig. S1 ). An overview of the key CFTR processes/modules included in CyFi-MAP is given to inform and guide the user to the map content (Fig.  1 ).

Modules available in the CyFi-MAP. wt- CFTR (left) and F508del- CFTR (right) modules include the key processes available included in the CyFi-MAP and provide a way to focus on a specific part of CFTR life cycle. Rescued F508del- CFTR (rF508del-CFTR) pathways are highlighted in yellow, indicating the processes elicited after chemical or temperature rescue of the mutant protein (see text for details).

Pathway inclusion strategy

We included information on CFTR interactors that was confirmed in a minimum of two published references. We focused on those that studied airway epithelial cells and used methodologies that allowed the detection of physical interaction between components (such as immunoprecipitation or nuclear magnetic resonance spectroscopy (NMR)). We also captured information on confidence and accuracy to each interaction in the map. Specifically, this step resulted in 296 research papers providing physical evidence between proteins and more than 1000 papers reviewed from PubMed (for more details on map curation see “ Methods ” section).

CyFi-MAP presents features that allow retrieving information visually such as (1) different types of interactions between the entities (i.e., activation, trafficking and inhibition—more details in Fig. S2 in Supplementary material), (2) the glycosylation (i.e., form B or C) and folding status (N-glycosylation) of CFTR , (3) the proteins that bind uniquely to the F508del- CFTR protein, (4) identification of eachlife cycle steps included—additional information in Supplementary material, and (5) cell organelles specific interactions represented through images, with the entities (i.e., proteins, complexes, ions, and others) adequately located in the biological compartments, differentiating between organelle lumen and the cytosol.

CyFi-MAP navigation

Map availability.

The source of these schemes are available at the map online repository ( https://cysticfibrosismap.github.io/ ) and the CyFi-MAP can be accessed online and explored interactively via the Molecular Interaction NetwoRks VisuAlization (MINERVA) 37 .

Online and interactive navigation

In the form of an interactive diagram via the MINERVA platform (Fig.  2 ), CyFi-MAP provides the capacity to easily follow CFTR interactions starting with its folding until degradation, with every intermediate step described (see supplementary material). The MINERVA platform allows easy navigation and exploration of the CF related molecular pathways available in CyFi-MAP. The user can also zoom in and find various details about proteins of interest (such as location of their interactions with other biological elements inside a cell, information on the protein name/alternative names and identification names in several databases such as Ensembl, human gene nomenclature, UniProt etc.; the annotation information is linked to the biological resources via direct urls). The user can also filter and extract information regarding the type of interaction through the edge colour (see Fig. S2 in Supplementary Material).

CyFi-MAP Github and MINERVA platform. In the image ( A ) is possible to see the GitHub page with the objective to contextualize this work and provide access to the interactive MINERVA platform. In image ( B ) and ( C ) are represented both submaps, wt- CFTR and F508del- CFTR respectively, where it is possible to navigate and explore the pathways and interactions included in CyFi-MAP, providing generic information about the proteins and components.

Content of the CyFi-MAP

The progression of CF disease is driven by the deregulation of multiple cell processes due to the loss of CFTR function. Hence, CyFi-MAP has greatly focused on the CFTR proteostasis network, as it encompasses alterations occurring on all those processes. Along with the map it is possible to observe several numbers which represent each step on the CFTR life cycle (a brief description of these is given in supplementary material) although the order indicated is not deterministic, serving only as an indication of all the processes in which CFTR is involved. The content included in the CyFi-MAP can be divided into four mains aspects: (i) CFTR synthesis and production, (ii) maintenance at the PM in the functional state, (iii) traffic and (iv) degradation.

CFTR synthesis and production

The folding of CFTR is highly regulated in the ER before the protein is allowed to proceed along the secretory pathway to the PM. This information is represented in the Folding module at both submaps hence, it is possible to visualize it side-by-side in the GitHub link.

Beginning with thewt- CFTR , folding starts with the nascent CFTR CFTR polypeptide chain being translocated to the ER membrane (step [1]), after which the N-glycosylation occurs (step [2]), a posttranslational modification during protein synthesis in the ER that is critical for PM expression and function (Fig.  3 A) 38 , 39 , 40 .In CyFi-MAP, the different glycosylation states of wt- CFTR are represented and possible to follow. N-glycosylation starts with the addition of oligosaccharide residues (glucose3-mannose9-N-acetylglucosamine2) to CFTR (Fig.  3 A, step [2]). At this step, several chaperones and co-chaperones bind to wt- CFTR to assist with folding. The folding process includes at least four ER quality control (ERQC) checkpoints that are involved in assessing CFTR correct folding 41 .As the process consists of the subsequent trimming of glucose, they are initially identified with three glucose (G3), the first checkpoint, before binding to calnexin ( CANX ) with only one (G1), the second checkpoint (Fig.  3 A, steps [3–6]) 42 .

Representation of folding at ER. Wt- CFTR ( A ) and F508del- CFTR ( B ) are subjected to the sequential ERQC checkpoints, indicated in black. On the wt- CFTR is possible to see the four checkpoints of the CFTR protein (in blue). In the case of F508del- CFTR is possible to see a bifurcation in the pathway, where it can be targeted to degradation or rescued, where it appears red at the cytosol. It is also indicated the beginning of the sumoylation degradation pathway. The proteins are represented in beige with the orange lines indicating the movement of the protein and the black lines indicating stimulation. The white boxes containing several proteins indicate a complex. Unique interactors to F508del- CFTR are represented in yellow.

F508del- CFTR is mostly targeted to degradation in the first checkpoint (Fig.  3 B, step [2]), hence the other ERQC checkpoints are only represented at the wt- CFTR submap There, is possible to follow the second checkpoint, where the protein enters the CANX cycle for additional rounds of refolding, the third checkpoint, where specific signals when exposed lead to ER retention, or the fourth checkpoint, with the recognition of an export motif to leave ER (Fig.  3 A, step [7] and step [8]) 43 . During these checkpoints, wt- CFTR can be recognized as misfolded and move to the degradation or achieves an incomplete glycosylated state known as form B, which allows it to proceed to Golgi. A more detailed description of these checkpoints is in the degradation chapter.

Although insertion in the membrane of ER and ERQC checkpoints that lead to degradation are identified as different steps, there is evidence that co-translational folding and degradation occur.

After CFTR folding has been successfully achieved, the protein is ready to proceed along the secretory pathway to the Golgi apparatus, where its oligosaccharide structure is further modified by multiple glycosylation events generating its mature form, known as C form, which will be transported to the PM 44 . F508del- CFTR , because is highly degraded at ER, from the moment is depicted outside this organelle acquires a dark red colour, representing a rescue protein rF508del- CFTR .

Maintenance at the plasma membrane in the functional state

After delivery to the PM, wt- CFTR is regulated at multiple levels namely: (1) PM stabilization, at specific PM sites; (2) activation/channel shut down, where phosphorylation/dephosphorylation cycles activate/inactivate the channels; (3) ion channels and transporters regulation, concerning the regulation of and by other PM proteins; and (4) endocytosis, in either CCVsor caveolae vesicles.

In contrast, rF508del- CFTR is characterized by (1) PM stabilization; (2) channel shut-down; and (3) endocytosis, with consequent degradation. The reduced number of modules at PM is representative of the instability, loss of function and accelerated endocytosis that characterize this mutated protein.

In the PM stabilization module, Postsynaptic density 95, disks large, zonula occludens-1 (PDZ) domain-containing proteins are the main characters, responsible for anchoring CFTR to the PM 45 . Mechanisms such as cytoskeletal activation are represented, which enable PM anchoring and tethering of wt- CFTR to the PM (Fig.  4 A, step [11]). TherF508del- CFTR stabilization module includes a lower number of interactions with PDZ proteins and acquisition of new interactions when compared to the wt- CFTR module (Fig.  4 B, step [5]).

PM stabilization in wt- CFTR ( A ) and F508del- CFTR ( B ) submaps. Wt- CFTR represented in blue is delivered to the apical PM in the C form, as indicated at the protein, and several proteins responsible for the anchoring to the PM bind. The lines show different types of interactions, where orange lines indicate traffic, being possible to see in chloride (Cl − ) transport across the membrane in orange. The black lines indicate binding/stimulation and the red one’s inhibition. F-actin and Arp2/3 are represented as complexes, with several proteins with a grey line separating them from the rest. In blue are represented 1-phosphatidyl1D-myo-inositol-4-phosphate (PIP) which by the action of Phosphoinositide Kinase, FYVE-Type Zinc Finger Containing ( PIKFYVE ) is converted to 1-phosphatidyl-1D-myo-inositol-4,5-bisphosphate (PIP2) with a blue line depicting synthesis. Although rF508del- CFTR reaches the PM, the binding to new proteins highlighted in yellow diminishes its stabilization and anchoring therefore accelerating its endocytosis and consequent degradation.

PDZK1 ( CAP70 ) is illustrated at PM in the wt- CFTR submap to be able to potentiate the CFTR chloride channel activity by cluster two CFTR molecules (wt- CFTR submap, step [12]) 46 (Fig.  4 A, step [12]).

Gating and channel shut down depict proteins involved in CFTR activation/inactivation (Fig.  5 ). CFTR is regulated through cAMP and phosphorylated by proteins such as protein kinase A ( PRKACA ) and Protein Kinase C Epsilon ( PRKCE ) 47 , 48 (Fig.  5 step [13]).This includes proteins such as β2-adrenergic receptor ( ADRB2 ) 49 , A2B receptor ( ADORAB2 ) 50 and adenylyl cyclase I ( ADCY1 ) 51 which participate in cAMP/PKA signalling therefore activating wt- CFTR and the transport of chloride.

PM pathways in wt-CFTR submap. ( A ) Gating where is possible to see adenosine 3,5-cyclic monophosphate (cAMP) role in Protein Kinase CAMP-Activated Catalytic Subunit Alpha (PRKACA) phosphorylation that leads to CFTR activation and transport of chloride across the membrane. Other proteins are involved such as Adrenoceptor Beta 2 (ADRB2), Protein kinase CK2 (formerly known as casein kinase II), Adenosine A2b Receptor (ADORA2B) and Protein Kinase C Epsilon (PRKCE). ( B ) Shut-down with the action of Lysophosphatidic Acid Receptor 2 (LPAR2), serine/threonine protein kinase complex (AMPK), Phospholipase C beta ½ (PLC beta ½) and Protein phosphatase 2 (PP2A) where the chloride transport is inhibited. ( C ) Transporters and Ion channel regulation, in which the proteins that regulate and are regulated by CFTR at the PM are represented with the interactions detected.

The Channel shut-down present in both submaps includes dephosphorylation of CFTR and the proteins involved in its triggering—such as protein phosphatases, receptors, phospholipases, and others 52 , 53 .

The transporters and Ion channel regulation module is only present at the wt- CFTR submap, and besides directly binding to wt- CFTR also allows to visualize PDZ proteins role as intermediates between them, maintaining the proteins in close proximity and leading to changes in their respective functions (Fig.  5 B/C, steps [16], [17] and [18]). Proteins such as Solute Carrier Family 26 Member 3 ( SLC26A3 , also known as DRA ), Solute Carrier Family 26 Member 6 ( SLC26A6 , also known as PAT1 ), Anoctamin 1 ( ANO1 , also known as TMEM16A ) and epithelial sodium channel ( ENaC ), were included in this module 54 , 55 , 56 .

In the secretory pathway, traffic is essential for all processes since folding/processing and function to degradation. CFTR traffic processes start by Coat Protein complex II (COPII) vesicles, responsible for its transport between ER and Golgi, from where wt- and rF508del- CFTR reach the PM (wt- CFTR submap, step [9]) 43 . rF508del- CFTR traffic between ER and Golgi is depicted through the COPII vesicles module with the same mechanisms as wt- CFTR (rF508del- CFTR submap, step [3]) 33 , 34 , 57 . Although this information is only supported by high-throughput research articles and with only one paper for each interaction with the mutated protein, it was one of the exceptional cases that were selected to CyFi-MAP given that is explained by the functional context provided in wt- CFTR . A list with the exceptions isin the additional information section of the supplementary material.

From this point, the endocytosis module depicts a wt- CFTR association with endocytic adaptors undergoing CCV-mediated and caveolae endocytosis (Fig.  6 A, steps [19, 20] respectively), whereas in rF508del- CFTR only the last one is depicted (Fig.  6 B, step [9]).

wt-CFTR ( A ) and rF508del-CFTR ( B ) endocytosis mechanisms of internalization from the PM. In the image A two types of internalization of wt-CFTR are present, through clathrin coated vesicles [19] and through caveolae vesicles [20]. As is represented, in the first, several proteins are involved, since the clathrin triskelion complex to cytoskeletal F-actin-MYO6 complex and other proteins assisting the process. In rF508del-CFTR, only the complex Caveolin 1 (CAV1)/ Caveolin 2 (CAV2) was found in this protein endocytosis [9], with assistance of Flotillin 2 (FLOT2).

CFTR is endocytosed and arrives at the sorting endosome from which wt- CFTR moves back to the PM, either directly—Recycling Module (Fig.  7 A, step [22], [23])—or through the Golgi—Golgi Module (Fig.  7 A, step [24])—or to degradation—Degradation Module (Fig.  7 A, step [26]). In these processes, several proteins of the Rab family are represented as they are essential for CFTR traffic 58 . In the case of rF508del- CFTR , at the sorting endosome, it is sent to degradation (Fig.  7 B, step [10]).

Sorting endosome in wt-CFTR ( A ) and F508del-CFTR ( B ) submaps. In the image ( A ), is possible to see wt-CFTR arriving at the sorting endosome [21] and the possible pathways to follow, either recycling [22], [23] and [24], or degradation [25] in orange lines. Bellow in image ( B ), rF508del-CFTR in red arrives at the endosome [10] in a complex with ubiquitin (represented with Ub) where several proteins target it to degradation directly, representing the low/absence of recycling of this protein. The proteins highlighted in yellow are unique to F508del-CFTR.

Degradation

During folding and processing, several quality control proteins target misfolded CFTR to degradation, therefore, in each of the organelles—ER and Golgi—there is a module called Degradation.

The four ERQC checkpoints can lead towt- CFTR degradationat the proteasome. In the case of F508del- CFTR , only the first ERQC checkpoint, called chaperone trap, is represented with the binding of four chaperones that strongly attach to the misfolded protein (i.e., Heat Shock Protein Family A ( Hsp70 ) Member 4 ( HSPA4 ), Heat Shock Protein Family A ( Hsp70 ) Member 8 ( HSPA8 ), DnaJ Heat Shock Protein Family ( Hsp40 ) Member A1 ( DNAJA1 ) and DnaJ Heat Shock Protein Family ( Hsp40 ) Member B1 ( DNAJB1 )) (F508del- CFTR submap, step [2A]) 42 , 59 . Furthermore, the F508del- CFTR presents a degradation pathway in contrast to wt- CFTR : the sumoylation (Fig.  3 B) 60 . At Golgi, CFTR degradation depicts its targeting to degradation via the lysosome. In the PM, the rF508del- CFTR is targeted to degradation by ubiquitinationas a consequence of the destabilization of the protein (F508del- CFTR submap, steps [7], [8] and [9]). There, proteins involved in its degradation at ER attach to itin the PM (i.e., HSPA8 , STIP1 Homology And U-Box Containing Protein 1 ( STUB1/CHIP ), Stress-Induced Phosphoprotein 1 ( STIP1 ) and others) 61 . At the sorting endosome, in both cells, the protein is targeted to degradation by lysosome/proteasome although with different interactors assisting (i.e., Tumor Susceptibility 101 ( TSG101 ), Hepatocyte Growth Factor-Regulated Tyrosine Kinase Substrate ( HGS ), Charged Multivesicular Body Protein 4B ( CHMP4B ), and others) bind uniquely to rF508del- CFTR (Fig.  7 B, step [10]) 62 .

CF is the most common life-threatening autosomal recessive disease in the Caucasian population 63 . Caused by an absent/dysfunctional CFTR channel that leads to an impairing balance of ions across the membrane, CF is characterized by affecting several organs, especially the lung 20 . CFTR seems to be involved not only in the transport of ions but also in the regulation of other channels, working in a dynamic network that modulates its activity 30 . There have now been more than 30 years of scientific discoveries with new milestones achieved every year in our understanding of intracellular interactions after CFTR loss of function that control the progression of this disease. The knowledge obtained over this research has enabled diagnosis and discovery of therapies that increased life expectancy 20 . Yet no final curative treatment has yet been developed for CF disease 64 , 65 , 66 .

The increasing data available on public databases lead to the improvement of tools to filter and extract relevant knowledge required for the discovery of therapeutic targets. With this need, disease maps were developed as a multilayer-readable network that allows representing increasingly complex and extensive information in an easily updatable manner 11 . The visual representation of CFTR key processes in a cell can act as a powerful tool to understand and share knowledge. In this work, we built the CyFi-MAP, a manually curated disease map of CFTR -related available information, as a resource that permits a deeper understanding and interpretation of the disease mechanisms. CyFi-MAP development is motivated by the absence of resources differentiating between wt- CFTR and its variants and has the objective of concentrating on a single free access resource the CF major hallmarks, representing the data scattered across different platforms/research papers in form of pathways and interactions. This tool was designed to be useful for CF scientists as a reference source to analyse previous knowledge and assist in the whole-organism level perspective as well.

CyFi-MAP included data: wt-CFTR versus F508del-CFTR submap

CFTR life cycle can be impacted at several steps, with the most common mutation, F508del- CFTR , subjected to ER retention and degradation when not rescued through low temperature or chemical compounds. In fact, rF508del- CFTR is characterized by barely reaching the PM under physiological conditions, presenting reduced chloride/bicarbonate transport after being rescued, as well as enhanced endocytosis and degradation, with consequent dysregulation of other PM proteins 53 , 67 . Due to F508del- CFTR incapability to achieve a competent intermediate, the protein becomes trapped in the first steps of ERQC and is mostly targeted for degradation as soon as the polypeptide is synthesized—indicated in CyFi-MAP by the absence of the others ERQC processes 42 , 43 , 68 , 69 . Furthermore, the location and function of CFTR at PM are affected in the case of rF508del- CFTR , visible by the lack of interactions and by the absence of key processes when compared with wt- CFTR in CyFi-MAP. rF508del- CFTR in CyFi-MAP lacks proteins involved in the activation of the channel and regulation of other channels and transporters at the PM. This is a consequence of its instability where unique proteins that interact with rF508del- CFTR , such as Calpain 1 ( CAPN1 ) and Calpain 2 ( CAPN2 ), play a role in its destabilization by impairing its binding to PDZ anchor proteins. Besides that, ubiquitination and subsequent targeting to endocytosis and degradation involve additional proteins such as Ring Finger And FYVE Like Domain Containing E3 Ubiquitin Protein Ligase ( RFFL ), TSG101 , HGS , CHMP4B and others that prevent the recycling to the PM 62 , 70 , 71 .

Some proteins (e.g. SLC9A3R1 , PDZK1 and others) involved in several roles along the CFTR life cycle (including stabilization, anchoring and function at PM) are present on the map more than once. PDZ proteins are essential elements also that act as intermediates that connect other channels with CFTR . Additionally, PDZK1 is found binding to two CFTR proteins, maintaining CFTR proteins functioning in close association.

Besides the conventional trafficking, unconventional secretion pathways have been described for membrane proteins such as CFTR and usually involve bypassing the Golgi, a route that was identified through blocking the conventional Golgi-mediated exocytic pathway 72 , 73 . Pathways such as this, were not included in this version of CyFi-MAP as they are unlikely to represent the cell in its physiological state. Notwithstanding, it can be helpful to provide a broader view of the possible interactions and they may appear in a future version of CyFi-MAP with less stringent criteria for inclusion.

CyFi-MAP expansion and future work

Given the fact that new data are generated continuously, and CF aspects are yet to be included, the CyFi-Map is constantly developed with support from the community and funding agencies.CyFi-MAP benefits from major features of the MINERVA platform via its online distribution: comments and suggestions from users with regard to changes in the map content (addition, removal, update) can be analysed directly by curators and addressed potentially in the map after further refinements. In this way, users can promote active discussions and knowledge exchange to build an increasingly accurate and continuously updated CF disease map. Notwithstanding, of specific interest as a future direction, is to include in CyFi-MAP the specific steps in CFTR processes targeted by compounds, depicting this way the specific target/mechanism where each of them acts. Additionally, we anticipate including a diagram focusing on the process description layer is anticipated of the CF molecular processes (e.g., the N-glycosylation of CFTR in ER) in order to provide a deeper understanding of such interactions. Furthermore, the creation of submaps representing other CFTR mutations would be relevant to study the molecular mechanisms affected.

Altogether, CyFi-MAP represents the first stable milestone into a robust and reliable CF knowledge base integrating information on key pathways involved in molecular pathophysiological CF mechanisms, based on curated literature and expert-domain-approval. CyFi-MAP offers an integrative and system-level view of CFTR knowledge. CyFi-MAP may support the interpretation of CF progression and may facilitate the development of novel therapeutic targets and strategies. In fact, a better understanding of CFTR mechanisms can not only assist in the design of improved therapies for CF but also identify factors that work in other lung diseases, such as COPD or disseminated bronchiectasis. The next steps can also involve the integration of the knowledge acquired using CyFi-MAP as a basis for mathematical models to generate new data through network inference, modelling and creation of new hypotheses to be tested.

CyFi-MAP construction

The development of the CyFi-MAP follows the disease map development protocol, using primarily Kondratova et al. and Mazein et al. 11 , 74 . Specifically, three main steps entail the construction of CyFi-CFTR (Fig.  8 ):

The first step consisted of searching relevant CFTR -related information, selecting a total of 297 research papers and more than 1000 reviewed articles. A complete list of publications consulted for the CyFi-MAP development is available in https://cysticfibrosismap.github.io/ . The CF disease hallmarks were obtained from peer-reviewed research papers, domain experts’ suggestions and advice, previously documented and validated pathways, and curated up-to-date databases (including Reactome 6 /KEGG 4 /MetaCore from Clarivate Analytics. Please see Content curation subsection for details). This task also involved the analysis of the collected pool of data, followed by the curation of the most relevant CFTR -related knowledge (Fig.  9 ).

The second step comprised the effective diagram building, assuring the correct level of detail and the most appropriate and aesthetically output, to guarantee that the resulting map is as readable and user-friendly as possible. The biological mechanisms representation follows the Systems Biology Graphical Notation (SBGN) notation and was built in the yEd Graph Editor using the SBGN Palette ( https://yed.yworks.com/support/manual/layout_sbgn.html ). The yEd Graph Editor is a freely available graph editor providing functionality to manage large-scale graphs including: (i) features that considerably facilitate the diagram drawing process such as friendly user interface, drawing guides, zooming on the diagram and easy application of specific aesthetics (e.g. same colour for nodes/ edges, curved connectors) to an individual or multiple elements; (ii) algorithms for automatic layout (details on using yEd to automatically layout SBGN-related diagrams are given in e.g. 75 ). The yEd Editor also incorporates the SBGN Palette that permits the direct representation of the SBGN-specific elements into the yEd inner GraphML format. After the CyFi-MAP was developed in yEd, we converted it into the SBGN standard format by using the ySBGN converter, (a bi-directional converter between the SBGN and yEd GraphML formats, available at https://github.com/sbgn/ySBGN ). Further, the CyFi-Map SBGN diagram was loaded to the MINERVA online platform. The organelle images (developed manually and expert-revised) aim to facilitate visualisation of the mechanisms at the top level; thus, special attention was given to the localization of the interactions in each organelle.

The third step in the construction of the CyFi-MAP was the map exploration via the MINERVA platform 37 . In a first approach, the construction focused on the creation of small organelle-specific maps, illustrating CFTR -relevant processes on those locations. The maps included CFTR interactions covering its intracellular and intraorganellar traffic. Later, these were improved upon by the addition of other, more widespread CFTR processes and pathways, which allowed a more effective integration of the existing data. The resulting cell-wide map is expected to be continuously evolving with user input and consistent expert curation. The map is available through the web platform MINERVA that provides interactive and exploratory features.

Workflow of CyFi-MAP construction. Starting by the research and curation of data contained in general and CFTR-specific databases and peer-reviewed research papers, CyFi-MAP was constructed based on domain experts’ suggestions and users’ comments. The organization of the curated data required the selection of a general format to be used throughout the platform, which determined the map assembly and content visualization. After the map was built, domain experts were once again consulted, in order to review and provide feedback on the accuracy of the representation of the disease mechanisms, as well as the usability of the platform.

CyFi-MAP curation process. The curation process developed presents 5 levels. 1st level filter the data to studies with proteins that interact directly with CFTR, meaning that only experimental techniques which confirmed a direct interaction were considered such as e.g. Immunoprecipitation, Surface Plasmon Resonance (SRP), and others. The 2nd level is related to the type of cell culture used in these experiments, focusing on human airway epithelial cells, although other cell types were included when described on review publications. In both levels, if the studie do not agree with the criteria is rejected. The 3rd level consist on finding the location of the interaction inside the cell (e.g. ER, Golgi, cytosol, PM) followed by the type of interaction (binding or inhibition). The 4th level confers confidence to the interaction, consisting on the search for publications that support the information. The 5th level confirms information related to the protein after being selected (e.g. does it belong to a complex? Which pathway does it belong to?). The protein is manually added to the yEd Graph Editor used to built CyFi-MAP with the name accordingly to the HGNC nomenclature.

The current version of CyFi-MAP has been manually curated by CF domain experts. To ensure a continuous updating of this resource, both regular expert verification of new information, as well as regular user input are deemed essential to achieve an accurate representation of current CF data. Constant feedback from cell biologists, biochemists, physiologists and bioinformaticians contributed to a comprehensive representation of the various layers of information.

Inside CyFi-MAP, each process comprises pathways that include proteins (as individual entities or as complexes) and different types of chemical species (ions and lipids) interacting among themselves. Nodes represent entities (i.e., proteins, ions or complexes) and edge colours correspond to processes (i.e., activation, inhibition, synthesis, or in some cases movement of entities inside the cell).

CyFi-MAP currently comprises 618 nodes and 420 edges, with 426 nodes and 307 edges in wt- CFTR and 216 nodes and 117 edges in F508del- CFTR . In total, entities presented at both submaps are classified into 193proteins, 25complexes, 5ions and 5 simple molecules in wt- CFTR and 98 proteins, 12 complexes, 1 ion, and 2 simple molecules in F508del- CFTR .

Content curation

The data used in CyFi-MAP was obtained by manual human search, curation and validation with domain experts in three main sources:

Pathway databases

The curation process started by reviewing previous attempts to summarize CF information in these signalling networks. Pathways from MetaCore (Clarivate Analytics), Reactome and KEGG were reviewed in order to analyse the pathway availability for the CF disease 6 , 76 . Major CF-related pathways were retrieved from these databases and confirmed in the literature for their accuracy.

The main hallmarks of CF were extensively searched in PubMed. As CFTR is the protein that plays a central role in the map, direct interactions with it were very carefully selected, following strict criteria. Considering that the lung is the most affected organ, the focus was on human airway epithelial cells studies. The massive number of CFTR reviewed articles and studies available were analysed and selected as particularly relevant; results obtained from essays with other relevant cell types (such as intestinal epithelial cells) were also included when validated by review papers, meaning they are accepted by the scientific research community. Priority in the selection process was given to the molecular mechanisms involving protein folding and traffic, as these are the main processes that are impaired in F508del- CFTR . Most studies regarding this mutant’s behaviour at the PM resulted from experiments on rF508del- CFTR , either chemically or temperature-dependent. The inclusion of information from proteomic studies was dependent on the functional context provided by already documented interactions. Although each direct interaction with CFTR on CyFi-MAP was confirmed in a minimum of two papers, some exceptions may apply such as information retrieved from recent articles (2018on) and protein interactions that were part of well-characterized pathways involved in CF referred in more than one peer-review research paper. An example of the last is the interaction between STX3 with CFTR , as only one research paper was found with a physical interaction although is mentioned in peer-review articles 77 and hence included as one interaction accepted by the scientific community.

Among the web resources used for data gathering, the most significant were GeneCards 78 , Stringdb 79 , Biogrid 80 , UniProt 81 and HGNC (HUGO Gene Nomenclature Comittee) 82 which were used to confirm the correct names of proteins/genes, their known function and their interactors. For each protein, the name was checked in HGNC. UniProt and Genecards were used to search for alternative names for the same protein so as to find the correct HGNC designation 78 , 81 , 82 . Often, although a protein complex is known to interact/participate in a CFTR process, the specific proteins that constitute that complex are not described in the original literature report. Accordingly, proteins reported in the literature to interact with CFTR as part of larger complexes were searched for in databases to find the protein components of the complex.

During this step, name disambiguation must be considered in order to find all data related to that protein and also to not repeat proteins. For instance, by looking for syntaxin 5, names such as Syn5 and STX5 are also available for the same protein. The same happens for Golgi Associated PDZ And Coiled-Coil Motif Containing protein, known as GOPC although other names such as CAL and FIG are referred to on research papers and used to retrieve as much information as possible.

Diagram building

CyFi-MAP was built using yEd graph editor ( https://www.yworks.com/ ) using the SBGN Palette, and the data was represented based on the SBGN standard 12 . This notation provides a knowledge representation language used in the illustration of molecular pathways and protein interactions as the standard notation for disease maps 11 . Presenting three languages that provide different types of knowledge illustration allow to adapt on the level of detail intended to be highlighted on the map, including the following layers: Activity Flow to depict interactions with process direction, Process Description detailed specific mechanisms, and Entity Relationships which describe the mechanisms without a sequential process 12 .

CyFi-MAP was implemented following the SBGN Activity Flow, in order to provide a compact, sequential and easy-read format or involving signalling pathways. This language is useful to represent the flow of information in biological sequences/pathways in a way that the information can still be captured for underlying mechanisms of unknown influence 12 .

Each subcellular organelle (ER, Golgi, endosome, etc.) were drawn manually and added to the map as a background image for graphical representation of the different subcellular compartments. Additionally, each interaction provides information on itself. Depending on the selected edge different types of interactions can be found on CyFi-MAP, namely (see Fig. S1 on supplementary material for more details): (1) activation, representing a normal binding, (2) synthesis, when an altered product is released, (3) trafficking, representing movement inside the map, and (4) inhibition, when the interaction inhibits a function.

Map exploration via the MINERVA platform

CyFi-MAP diagrams are available in the platform MINERVA accessible through GitHub ( https://cysticfibrosismap.github.io/ ). The project description and key processes (shown side-by-side, represented through images to allow the comparison between wt- CFTR and F508del- CFTR submaps on the CyFi-MAP) are given on the website. Starting at the cell level, it is possible to identify the main differences between the submaps (Fig.  1 ). This view is relevant in order to compare the cells in presence of the two proteins since wt- CFTR is transported across the secretory pathway to the PM and endocytosed to be either degraded or recycled back to the PM, whereas most F508del- CFTR is retained at the ER from where it is sent for degradation. This impairment leads to the so-called ‘CF pathogenesis cascade’, which does not occur for wt- CFTR . In the cell level view, it is possible to observe these features, allowing the extraction of relevant knowledge.

Additionally, wt- CFTR and F508del- CFTR submaps were divided into modules, each representing key processes of its life cycle in order to guide the navigation through the map. To compare information in both submaps, images placing the modules folding, stabilization and sorting side-by-side are available. Mutation-specific proteins are highlighted in a different colour to emphasize differences between wild-type and mutated phenotypes.

The interactive web platform MINERVA allows accessing an interactive CyFi-MAP to navigate and explore its molecular networks. This tool provides automated content annotation, direct feedback to content curators and SBGN-compliant format 83 . Navigation in CyFi-MAP is similar to navigation in Google Maps being possible to through MINERVA search elements that are highlighted by markers and also retrieve additional information on each element on the panel on the left side, presenting several identifications names using HGNC and UniProt as sources.

The zoom feature allows a high-level view of the intracellular organelles and a close view inside each providing easier access to the complex and extensive information it contains. Every CFTR interaction represented in the CyFi-MAP is validated by PubMed references. The user can curate the data by commenting, given they provide the respective reference as well.

All suggestions will be analysed by curators and CF domain experts to maintain CyFi-MAP quality and accuracy.

The user can contribute to CyFi-MAP by adding comments with questions, corrections or additions to the map. These will be visible to other users and developers. To add a comment to CyFi-MAP during navigation, right-click on the specific location and choose to add a comment. It is possible to choose a specific identity to link the comment, such as the protein or reaction, or to remain ‘general’, which will link the comment to the location the user chooses. The remaining fields allow the user to fill in the name and email in order to facilitate communication with the developers and to clarify any questions that may emerge. Last, there will be a box where comments can be added (Fig. S2 ). Any supporting information provided will be helpful to incorporate the changes into the map. After sending the comment, it will not be possible to correct it and it will be visible on the map publicly. Details on adding user’s comments in the underlaying MINERVA platform are given at https://minerva.pages.uni.lu/doc/user_manual/v15.0/index/#add-comment .

The CyFi-MAP allow exploring the map with and without seeing the comments provided by the users by clicking on the checkbox Comments above in the map toolbar. These comments will allow the map users to benefit from the domain knowledge and expertise of researchers and to collect valuable information for the research community. All suggestions will be analysed by curators and CF domain experts in agreement with the pre-established curation process to maintain CyFi-MAP quality and accuracy.

Data availability

The CyFi-MAP is available at https://cysticfibrosismap.github.io/ .

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Acknowledgements

The authors would like to thank the domain experts that participated in stimulating discussions and valuable feedback and to the colleagues from FunGP—Functional Genomics and Proteostasis who tested CyFi-MAP and provided valuable suggestions. Work in MDA lab is supported by UIDB/04046/2020 and UIDP/04046/2020 centre grants (to BioISI) from FCT/MCTES Portugal. CP was recipient of fellowship SFRH/PD/BD/131405/2017 and through funding of LASIGE Research Unit, ref. UIDB/00408/2020 and ref. UIDP/00408/2020. AM and IB were supported in part by the Innovative Medicines Initiative Joint Undertaking under Grant Agreement No. IMI 115446 (eTRIKS), resources of which are composed of financial contributions from the European Union’s Seventh Framework Programme (FP7/2007–2013) and EFPIA companies.

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Contributions

C.P.—conceptualization; data curation; formal analysis; investigation; methodology; original draft preparation; review and editing; validation. A.M.—conceptualization; methodology; review and editing; supervision. C.F.—review and editing. M.G.—review and editing. K.K.—review and editing. M.O.—MINERVA online version. I.B.—MINERVA online version. M.D.A.—conceptualization; funding acquisition; resources; review and editing supervision. A.O.F.—conceptualization; data curation; methodology; resources; review and editing; supervision; validation. All authors have read and agreed to the published version of the manuscript.

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Correspondence to Andre O. Falcao .

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Pereira, C., Mazein, A., Farinha, C.M. et al. CyFi-MAP: an interactive pathway-based resource for cystic fibrosis. Sci Rep 11 , 22223 (2021). https://doi.org/10.1038/s41598-021-01618-3

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Introduction

Instructional context, assessment objectives, case study background, student activities, student survey, assessing student conceptions of protein synthesis with a case study in crispr and de-extinction.

KADEE G. RUTKOWSKE ( [email protected] ) is an undergraduate learning assistants at Michigan State University and participated equally in the implementation, analysis, and writing stages of this research project.

JACOB N. WILLIS ( [email protected] ) is an undergraduate learning assistants at Michigan State University and participated equally in the implementation, analysis, and writing stages of this research project.

ANDREA M.-K. BIEREMA ( [email protected] ) is an academic specialist at Michigan State University.

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Kadee G. Rutkowske , Jacob N. Willis , Andrea M.-K. Bierema; Assessing Student Conceptions of Protein Synthesis with a Case Study in CRISPR and De-extinction. The American Biology Teacher 1 September 2022; 84 (7): 415–421. doi: https://doi.org/10.1525/abt.2022.84.7.415

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Scientific modeling is a practice that we use frequently in our undergraduate biomedical applications course for nonscience majors. We use case studies in which students apply course concepts to create cause-and-effect models. In this article, we describe a case study assessment on protein synthesis that examines the use of CRISPR to bring back the mammoth (i.e., de-extinction). Students learn about protein synthesis throughout the course and work on various case study scenarios to apply those concepts. Their final assessment is a team project to illustrate how protein synthesis is influenced by gene editing, including gene expression and its regulation, transcription, translation, protein structure and function, and the ultimate impact on an organism’s phenotype. Although we use this case study as an assessment, it is also appropriate as a class activity in which students practice modeling the CRISPR gene-editing system.

Calls for undergraduate education reform recommend focusing courses on scientific practices, crosscutting concepts, and core ideas ( Cooper et al., 2015 ). To meet these calls, our undergraduate students develop scientific models (a scientific practice) to explain how molecular structures and processes influence an organism’s physiology and appearance (core ideas from HS-LS1 in NGSS, 2013 ). Prior to each modeling activity, students prepare by viewing informative videos with an associated open-note quiz that covers basic concepts. During class, students apply those concepts to novel cases, such as how gene expression regulation causes stickleback fish to develop—or not develop—spines (activity modified from HHMI Biointeractive, 2011 ). For each unit of the course, students repeat this process on related concepts for a few class periods and then apply all concepts from the unit in a modeling assessment regarding a single case study.

In class, student teams use case studies as context in which to apply the concepts that they learned during the preparation phase .

This course focuses on protein synthesis and gene regulation, such as how enhancers influence gene expression, and during the last unit of the course, students apply those concepts to illustrate how various biotechnologies work and influence gene expression. The assessment described in this article focuses on an emerging gene-editing biotechnology: CRISPR. First, we describe CRISPR and the associated assessment case study on mammoth de-extinction. We explain the team-based modeling assessment activity and present students’ performance results.

The course is an undergraduate introductory biology course offered to nonscience majors and enrolls 100–150 students. We implemented this assessment to determine students’ knowledge of protein synthesis. We used a flipped-style approach in which students complete short readings and preparatory quizzes prior to each class period. During class, students work in teams consisting of three to four members. We created these teams using a team-maker software called CATME (Purdue University, www.catme.org ), which creates a student survey and places students in teams based on the results of that survey. The survey includes questions about demographics, team preferences, and expected effort in the class. We have teams composed of individuals with similar team preferences and expected class effort. Teams are diverse in the demographic information that we ask about (e.g., gender) but we also ensure that no one is by themselves within a team (e.g., not having one person that identifies as female with teammates that all identify as male).

In class, student teams use case studies as context in which to apply the concepts that they learned during the preparation phase—for most tasks, students develop models that explain the underlying mechanisms relevant to the case study situation. While students work, the instructional team, consisting of the professor and two undergraduate learning assistants who previously took the course, walk around to answer student questions, confirm that all students are working, and ask questions to further their comprehension.

The course is structured into five units, each focusing on a group of related concepts. Unit 1 starts with an introduction to the nature of science, and Unit 2 focuses on information literacy concepts. The remaining units focus on various biological concepts: Unit 3 deals with proteins and cell communication, Unit 4 covers gene expression, and Unit 5 focuses on genetic applications, including CRISPR. For Units 1–4, students complete individual in-class exams using case studies; the Unit 5 exam—the subject of this article—is a team-based assessment where students are asked to apply protein synthesis concepts learned and practiced during Units 3 and 4 to a case study involving CRISPR, which is the focus of this article. Each exam consists of a mix of forced-choice and constructed-response questions, including developing models.

Units 3 and 4 focus on protein synthesis at a fairly broad level due to the introductory level of the course. We begin by introducing how the sequence of amino acids affects the structure of the protein. Students are given an amino acid chart with associated charges to create a model of three similar but different proteins. They develop models of the primary structure (i.e., the list of amino acids) and the tertiary level, but for our purposes, we focus on a two-dimensional structure. That is, students consider how charges and disulfide bonds create folds in the peptide. This demonstrates how single substitutions of different charged amino acids can alter protein structure and subsequent function. During this activity, students are also introduced to how the similarities of protein structure and function translate to evolutionary relatedness between species. In a higher-level class, instruction should consider how charges, as well as polarity, affect the three-dimensional structure of a protein and to what extent these changes affect the function. For the purposes of this course, we considered only that it would change the function, not how specific changes in shape may cause specific functions or lack of functions (e.g., how a change in keratin, hair protein, can cause the hair to appear curly).

To understand protein synthesis, we first describe the anatomy of a DNA molecule, including enhancers, promoter, protein-coding region (including codons), and termination sequence. Multiple case studies are used throughout to help students understand how the anatomical structures relate to protein synthesis. After students practice drawing DNA molecules and protein structures, we have students model protein synthesis. Given novel case studies, they draw the two main processes of protein synthesis: transcription and translation (spending an entire class period on each process). While building translation models, students are expected to understand the roles of mRNA, tRNAs and ribosomes. Finally, we introduce how protein synthesis can regulate gene expression using enhancers or alternative splicing. All these concepts are wrapped up through the final assessment, which includes building a CRISPR model.

Our assessment uses CRISPR as a grounding element to measure students’ understanding of protein synthesis. The following are the assessed objectives, which incorporate concepts from the previous units, including amino acid properties, protein structure and function, transcription, translation, gene expression and regulation (or regulation of gene expression), and mutations.

Develop and use a scientific model based on amino acid properties to test and illustrate protein structure and function.

Make appropriate inferences of protein structure and function models.

Predict the extent of similarity of protein structures based on the extent of similarity in protein functions and evolutionary relatedness.

Identify the switches, promoter, and transcription starting regions on a DNA molecule.

Describe how activators, transcription factors, and polymerase proteins are used in transcription.

Predict how mutations in various parts of a DNA sequence influence protein expression.

Explain how different cell types perform different functions.

Describe the roles of mRNA, tRNAs, and ribosomes in protein translation.

Describe how translation starts and ends, including the functions of start and stop codons.

Describe the anatomy of a tRNA.

Interpret a codon–amino acid chart.

Describe examples of protein synthesis regulation.

Create a model to explain how cells regulate gene expression using switches or exons and introns.

In doing this assessment, students learn about CRISPR and meet the following objectives. These objectives, though, were not part of the assessment, so students were welcome to ask questions during class to help in their understanding of these objectives.

Identify the molecular steps and their variations in CRISPR.

Develop a scientific model that illustrates how CRISPR works in the context of a novel case study.

Apply concepts of genetic mutations, protein synthesis, gene expression, regulation, and protein structure and function to CRISPR.

Scientists modify genes in a variety of ways, including cloning (inserting DNA from an individual into a host egg cell of another), reconstructing the DNA chemically (if the full sequence is available), and using CRISPR (editing specific sequences). The basis of the technology is that short RNA sequences guide the protein Cas9 to its matching DNA sequences where Cas9 binds and cuts the target gene ( Broad Institute, 2018 ). Once the DNA is cut, researchers allow the cell’s natural repair mechanisms to repair the genetic material; this creates a random mutation in the gene, which most often makes it nonfunctional ( National Institutes of Health, 2020 ). Alternatively, scientists can insert a new piece of DNA.

One possible application of CRISPR is de-extinction, which has recently come to light as a highly debated scientific technology due to ethical, social, and ecological concerns in the media as well as among scientists at public events and even at special symposia at academic conferences ( Novak, 2018 ). The International Union for the Conservation of Nature guidelines define de-extinction as generating a new species that is altered to serve a similar ecological function as the original species ( Novak, 2018 ). Because an extinct species lineage can never be fully recovered, its ecological function cannot be identical to the original, extinct species ( Novak, 2018 ).

Within the mass media, one of the most commonly discussed candidate species for de-extinction is the woolly mammoth. Mammoths likely had a huge impact on the ecosystem by maintaining grasslands in areas that later transformed to woodlands after their extinction ( McCauley et al., 2016 ). Given that woolly mammoths went extinct thousands of years ago, the samples of DNA sequences that scientists are able to extract are contaminated with DNA from other organisms such as bacteria (e.g., CCGB, 2013 ), so they need to use a “template” (or surrogate species) for developing the entire genome. Therefore, it is likely that CRISPR is the most applicable tool in comparison to DNA reconstruction or cloning, should scientists ever decide to bring back the woolly mammoth. CRISPR is used to edit the genes of the surrogate’s embryo so that it closely resembles a woolly mammoth, and then the surrogate parent carries the embryo to term. Therefore, the surrogate species is the link to generate the new species of mammoths. The closest genetic living relative of the mammoth is the Asian elephant ( Rohland et al., 2007 ), and therefore, it is the most likely surrogate species. Again, the extinct mammoth species will likely never be recovered, but CRISPR makes it possible to use the partial DNA available from the extinct mammoth and DNA of its closest living relative, the Asian elephant, to create a new species capable of serving a similar ecological function as the woolly mammoth. Because Asian elephants live in a very different environment from the woolly mammoth, there are a few key traits that would likely need to change, including the blood’s ability to release oxygen at low temperatures, subcutaneous fat for insulation and storage, thick hair, and smaller ears and tail (see Lynch et al., 2015 , for a review).

This activity is an assessment that applies protein synthesis concepts from throughout the semester to a novel case study; that is, how an edit to a gene via CRISPR may change how that gene is synthesized and/or how that edit affects the organism. In this case, an Asian elephant’s genome is modified and inserted into an embryo so that the embryo results in mammoth-like Asian elephant. To prepare for this assessment, students create two in-class models that apply course concepts to other biotechnologies (i.e., genetically modified organisms and gene therapy) and complete a homework assignment that introduces students to how CRISPR works. They were introduced to the general idea of CRISPR and its controversial issues earlier in the semester by reflecting on the following TEDTalks:

“Changing the Human Story with CRISPR-Cas9”

“The Ethical Dilemma of Designer Babies”

“What You Need to Know About CRISPR”

The first video gives a very positive position on CRISPR by an undergraduate researcher, including how easy it is to use. Having a video by an undergraduate was purposely selected to give students a more relatable speaker. The second video, by a biologist, focused on a potential ethical dilemma of CRISPR, and thereby providing a negative view on CRISPR. The third video, by an established CRISPR researcher, describes both positive and negative concerns of CRISPR.

Preparation Case Studies

In preparation for the CRISPR case study, the two class periods before the assessment involve practicing modeling how a biotechnology works and how the concepts from the course help explain how that biotechnology influences an organism’s phenotype: one case is on genetically modified foods, and the other is on gene therapy. The GMO case study on AquAdvantage salmon prepares students by having them model the genetic process of the normal Atlantic salmon and the AquAdvantage salmon. Then students use this information to explain how the cell types are different. The gene therapy case study on cystic fibrosis has students model protein synthesis and challenges students to apply concepts of protein synthesis, gene expression regulation, and the effects of gene therapy on protein structure and function. Students practice using these activities to piece together the process in which an organism can be edited using biotechnology techniques.

Preassessment Homework

To prepare for the CRISPR modeling assessment, students view the following series of videos and participate in an interactive that shows how CRISPR works:

Rachel Haurwitz (2016), “CRISPR: Editing Out Genetic Instructions,” https://www.youtube.com/watch?v=wktwXGAbP_Q

McGovern Institute (2014), “Genome Editing with CRISPR-Cas9,” https://www.youtube.com/watch?v=2pp17E4E-O8

Seeker (2016), “What Is CRISPR and How Could It Edit Your DNA?,” https://www.youtube.com/watch?v=SyAo51IYgUw

Yourgenome (2016), “What Is CRISPR-Cas9?,” https://www.yourgenome.org/facts/what-is-crispr-cas9

HHMI Biointeractive (2019) , “CRISPR-Cas9 Mechanism & Applications,” https://www.biointeractive.org/classroom-resources/crispr-cas-9-mechanism-applications

Then students complete a multiple-choice quiz that addresses the main components and steps of CRISPR as a homework assignment that is graded based on correctness.

In-Class Modeling Assessment

For the modeling assessment, students answer the question “How can CRISPR be used to modify DNA in the Asian elephant to produce protein found in a mammoth?” In the worksheet, students first identify natural ways in which two species can create similar but different proteins. In our class, students often explored this question (e.g., a mutation causing a deletion of a switch that had turned on the gene in a specific cell type) and had to address it in writing as they learned about additional mechanisms. Without this background, though, the question may result in confusion, as students may think it is just referring to CRISPR rather than which changes in a gene result in different proteins.

Students use the concepts that they learned throughout the semester to create a model explaining how an edit in a gene of an Asian elephant embryo affects protein synthesis and results in a genetically modified elephant that is similar to a woolly mammoth (see the worksheet in Figure 1 for the specific questions that the model must address).

Student worksheet. Part 1 prepares students to create the model, and Part 2 is the modeling activity.

While students work on the model, the instructor walks around, providing guidance to students who request assistance in understanding how the model should explain certain concepts about gene editing and tying all the processes together. The students’ most common questions are about the mechanism of CRISPR and how it works. The instructor answers any questions regarding CRISPR and de-extinction but not questions on how protein synthesis works, because the students are being assessed on that knowledge. If a student simply asks if something is correct, the instructor refers them to the grading rubric.

Model Evaluations

We graded students’ models using an analytic rubric ( Table 1 ). Overall, students’ models met the exemplary level in the rubric and demonstrated an understanding of protein synthesis ( Figure 2 ). Most students correctly transcribed a DNA sequence to an mRNA sequence and translated it into an amino acid sequence (see Figures 3 and 4 for examples and Supplement Material, available with the online version of this article, for an exemplar model developed by the authors). Several models lacked a protein shape—that is, how the charges of the amino acids influence the shape of the protein ( Figure 3 ). It is unclear, though, if this trend was due to a lack of understanding of the concept or a lack of explicit instructions. The rubric ( Table 1 ) used for this analysis was revised from the original grading rubric to explicitly state that exemplary work includes specific amino acids in the predicted shape, based on their characteristics, and models that just include lines are categorized as needing improvement. The original grading rubric, which students could access during the assessment, did not contain those instructions, but this instruction was included in the worksheet and practiced previously during class.

Rubric used to assess students’ models. Bold and italicized text are phrases used in this assessment but not the original grading of the models.

Heat map illustrating evaluation results of all student models. Each row represents one model, and each column is a criterion from the rubric (Figure 2). Shading aligns with the levels in the rubric: light gray is exemplary, and dark gray is not evident (see key).

An example of a student model that received a perfect score in the course but not in this analysis (because it does not include specific amino acid charges impacting the protein’s shape). Starting from the top of the model and working counterclockwise, this illustrates how the guide RNA and CAS-9 function and cause a mutation that can impact protein formation.

An example of a student model that received a perfect score on the assessment. Starting from the top of the model, it illustrates the predicted DNA difference between an Asian elephant and mammoth. The bottom of the model illustrates variation in protein shape affects the phenotype (i.e., thicker or thinner hair).

Most students modeled CRISPR to our expectations. As seen in the rubric, they focused on explaining the role of the guide RNA and Cas-9. Most models missed the steps between cutting the DNA and inserting a new sequence; for instance, Figure 4 includes an explanation that a DNA sequence is inserted but none about how this insertion occurs. This was not a surprise because it was not explicitly included in the instructions—just that they needed to show how CRISPR works. Two models described that “replacement DNA (mammoth DNA) is provided by scientists,” and seven models mentioned the cell’s repair mechanism (e.g., “the cell’s repair systems stitch the replacement DNA into the genome”). Both of those concepts were not explicitly required for this assessment but were explained in the preparation materials. Additionally, most models included the unlikely case of a guide RNA being just a few bases long (e.g., Figure 3 ). For our objectives, this was not an issue, but for biology-major courses, this could create a critical misconception that a single base pair can be edited.

Since the Asian elephant lives in a warmer climate, it has evolved to not use the protein that releases blood oxygen at low temperatures. The amino acid sequence for the protein can still be in the DNA, but there is no switch [enhancer] that activates it. By using CRISPR-CAS9 to insert the switch, the Asian elephant can produce the protein again .

Toward the end of the first semester of using this assessment, students anonymously completed an internal evaluation survey. This instrument is used in courses within our department to assess students’ perceptions of each course’s activities and pedagogical strategies. It contains a core set of questions used in every class and a set of questions specific to the course, such as certain class activities. This was the first semester that the survey was used in the department, and so the primary goal was to test the instrument. We took advantage of this opportunity by adding a question about the CRISPR modeling assessment. Students completed it during class for extra credit and were made aware that the instructor could not access the results until after submitting final grades. Of the 138 students enrolled in the course, 132 students completed at least the multiple-choice portion of the survey (96%), and 120 students also answered most of the open-response questions (87%).

One question asked to rate the helpfulness of the CRISPR modeling assessment. On a five-point Likert scale in which 5 meant that the assessment greatly helped and 1 meant that it did not help, the average score was 3.81 ( n = 132), with 29% of students selecting 5 and 3% choosing 1 ( Figure 5 ).

Likert scale results for the survey question “Please rate the extent to which the CRISPR activity helped your learning” ( n  = 132, 96%).

The class is taught so that you work in groups and work on problems of a specific topic with the members of your group to help solve it. For example … we had to make a CRISPR model working with the members of our team, using knowledge that we each had to help complete it .

If they were well explained and the online activity was actually good prep for it, then they were very helpful. Other times, there was way too big of a jump from the content of the online module and the class activity, and since there is no instructional time in the course, I very often felt lost during class .

This concern alludes to the importance of having pre-activity materials that prepare students for upcoming class periods. For the second semester, we added the interactive tutorial from HHMI Biointeractive, “CRISPR-Cas 9 Mechanism & Applications,” to the homework, and the instructor noticed nearly all teams had at least one student opening and going through the interactive while working on their CRISPR model.

This modeling assessment gave students a chance to not only learn about CRISPR, an emerging biomedical application, but also apply concepts of protein synthesis, gene regulation, and mutations to a novel case. Most recently, we used this assessment as a regular in-class activity, and then the assessment had students create a similar CRISPR model for another topic that we randomly assigned to each student, such as HIV treatment or crop longevity. They completed these activities on their own time, and the models are similar to the assessment models analyzed in this article.

The dissemination of these results was approved by the institution’s review board (Study ID: STUDY00003803).

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Cystic fibrosis occurs when the cystic fibrosis transmembrane conductance regulator (CFTR) protein is either not made correctly, or not made at all. By understanding how the protein is made, scientists have been able to develop treatments that target the protein and restore its function.

• The cystic fibrosis transmembrane conductance regulator (CFTR) protein helps to maintain the balance of salt and water on many surfaces in the body, such as the surface of the lung.
• The CFTR protein is a particular type of protein called an ion channel. In the lung, the CFTR ion channel moves chloride ions from inside the cell to outside the cell.

Researchers are still trying to learn more about the structure of the CFTR protein so that they can find new and better ways to help improve the function of the protein in people with CF.

The cystic fibrosis transmembrane conductance regulator (CFTR) protein helps to maintain the balance of salt and water on many surfaces in the body, such as the surface of the lung. When the protein is not working correctly, chloride — a component of salt — becomes trapped in cells. Without the proper movement of chloride, water cannot hydrate the cellular surface. This leads the mucus covering the cells to become thick and sticky, causing many of the symptoms associated with cystic fibrosis.

To understand how mutations in the CFTR gene cause the protein to become dysfunctional, it is important to understand how the protein is normally made, and how it helps to move water and chloride to the cell surface.

What Are Proteins?

Proteins are tiny machines that do specific jobs within a cell. The instructions for building each protein are encoded in DNA. Proteins are assembled from building blocks called amino acids. There are 20 different amino acids. All proteins are made up of chains of these amino acids connected together in different orders, like different words that are written using the same 26 letters of the alphabet. The DNA instructions tell the cell which amino acid to use at each position in the chain to make a specific protein.

The CFTR protein is made up of 1,480 amino acids. Once the CFTR protein chain is made, it is folded into a specific 3-D shape. The CFTR protein is shaped like a tube that goes through the membrane surrounding the cell, like a straw goes through the plastic top on a cup.

What Does the CFTR Protein Do?

The CFTR protein is a particular type of protein called an ion channel. An ion channel moves atoms or molecules that have an electrical charge from inside the cell to outside, or from outside the cell to inside. In the lung, the CFTR ion channel moves chloride ions from inside the cell to outside the cell. To get out of the cell, the chloride ions move through the center of the tube formed by the CFTR protein.

Once the chloride ions are outside the cell, they attract a layer of water. This water layer is important because it allows tiny hairs on the surface of the lung cells, called cilia, to sweep back and forth. This sweeping motion moves mucus up and out of the airways.

How Do Problems With the CFTR Protein Cause CF?

In people with CF, mutations in the CFTR gene can cause the following problems with the CFTR protein:

• It doesn't work well
• It isn't produced in sufficient quantities
• It is not produced at all

When any of these problems occur, the chloride ions are trapped inside the cell, and water is no longer attracted to the space outside the cell. When there is less water outside the cells, the mucus in the airways becomes dehydrated and thickens, causing it to flatten the cilia. The cilia can't sweep properly when thick, sticky mucus weighs them down.

Because the cilia can't move properly, mucus gets stuck in the airways, making it difficult to breathe. In addition, germs caught in the mucus are no longer expelled from the airway, allowing them to multiply and cause infections. Thick mucus in the lungs and frequent airway infections are some of the most common problems people with CF face.

Researchers Are Still Studying the Basic Structure

Because the 3-D shape of CFTR is so complex, it was not until early 2017 that the first high-resolution pictures were developed. These pictures have given researchers important clues about where drugs bind the protein, how they affect its function, and how to develop new CF therapies. In the future, pictures showing the protein in an “open” position, where salt can move through, will be even more helpful to researchers developing new CF therapies.

CF Genetics: The Basics Article | 6 min read

Types of CFTR Mutations Article | 9 min read

Restore CFTR: Exploring Treatments for Rare and Nonsense Mutations Article | 8 min read

Find Out More About Your Mutations Article | 3 min read

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