Ribosomal Frameshifting Selectively Modulates the Assembly, Function, and Pharmacological Rescue of a Misfolded CFTR Variant.

The cotranslational misfolding of the cystic fibrosis transmembrane conductance regulator chloride channel (CFTR) plays a central role in the molecular basis of cystic fibrosis (CF). The misfolding of the most common CF variant (ΔF508) remodels both the translational regulation and quality control of CFTR. Nevertheless, it is unclear how the misassembly of the nascent polypeptide may directly influence the activity of the translation machinery. In this work, we identify a structural motif within the CFTR transcript that stimulates efficient -1 ribosomal frameshifting and triggers the premature termination of translation. Though this motif does not appear to impact the interactome of wild-type CFTR, silent mutations that disrupt this RNA structure alter the association of nascent ΔF508 CFTR with numerous translation and quality control proteins. Moreover, disrupting this RNA structure enhances the functional gating of the ΔF508 CFTR channel at the plasma membrane and its pharmacological rescue by the CFTR modulators contained in the CF drug Trikafta. The effects of the RNA structure on ΔF508 CFTR appear to be attenuated in the absence of the ER membrane protein complex (EMC), which was previously found to modulate ribosome collisions during preemptive quality control of a misfolded CFTR homolog. Together, our results reveal that ribosomal frameshifting selectively modulates the assembly, function, and pharmacological rescue of a misfolded CFTR variant. These findings suggest interactions between the nascent chain, quality control machinery, and ribosome may dynamically modulate ribosomal frameshifting in order to tune the processivity of translation in response to cotranslational misfolding.

Proteostasis Landscapes of Selective versus Poorly Responsive CFTR Variants Reveals Structural Vulnerabilities to Correction.

Cystic Fibrosis (CF) is a lethal genetic disorder caused by variants in CF transmembrane conductance regulator (CFTR). Many disease variants are treatable with corrector compounds, which enhance the folding and trafficking of CFTR. However, correctors fail to elicit a response for every CFTR variant. Approximately 3% of persons with CF harbor poorly responsive CFTR variants. Here, we reveal that a group of poorly responsive variants overlap with selectively responsive variants in a critical domain interface (nucleotide-binding domain 1/intracellular loop 4 - NBD1/ICL4). Affinity purification mass spectrometry proteomics was used to profile the protein homeostasis (proteostasis) changes of CFTR variants during corrector treatment to assess modulated interactions with protein folding and maturation pathways. Responsive variant interactions converged on similar proteostasis pathways during correction. In contrast, poorly responsive variants subtly diverged, revealing a partial restoration of protein quality control surveillance and a capacity to correct some mutations. Computational structural modeling showed that corrector VX-445 failed to confer enough NBD1 stability to poorly responsive variants. NBD1 secondary stabilizing mutations rescued poorly responsive variants, revealing structural vulnerabilities in NBD1 required for treating poor responders. Our study provides a framework for discerning the underlying protein quality control and structural defects of CFTR variants not reached with existing drugs. These insights can help expand therapeutics to all susceptible CFTR variants to enhance personalized medicine efforts.

Benchmarking AlphaMissense pathogenicity predictions against cystic fibrosis variants.

Variants in the cystic fibrosis transmembrane conductance regulator gene (CFTR) result in cystic fibrosis-a lethal autosomal recessive disorder. Missense variants that alter a single amino acid in the CFTR protein are among the most common cystic fibrosis variants, yet tools for accurately predicting molecular consequences of missense variants have been limited to date. AlphaMissense (AM) is a new technology that predicts the pathogenicity of missense variants based on dual learned protein structure and evolutionary features. Here, we evaluated the ability of AM to predict the pathogenicity of CFTR missense variants. AM predicted a high pathogenicity for CFTR residues overall, resulting in a high false positive rate and fair classification performance on CF variants from the CFTR2.org database. AM pathogenicity score correlated modestly with pathogenicity metrics from persons with CF including sweat chloride level, pancreatic insufficiency rate, and Pseudomonas aeruginosa infection rate. Correlation was also modest with CFTR trafficking and folding competency in vitro. By contrast, the AM score correlated well with CFTR channel function in vitro-demonstrating the dual structure and evolutionary training approach learns important functional information despite lacking such data during training. Different performance across metrics indicated AM may determine if polymorphisms in CFTR are recessive CF variants yet cannot differentiate mechanistic effects or the nature of pathophysiology. Finally, AM predictions offered limited utility to inform on the pharmacological response of CF variants i.e., theratype. Development of new approaches to differentiate the biochemical and pharmacological properties of CFTR variants is therefore still needed to refine the targeting of emerging precision CF therapeutics.

Benchmarking AlphaMissense Pathogenicity Predictions Against Cystic Fibrosis Variants.

Variants in the cystic fibrosis transmembrane conductance regulator gene (CFTR) result in cystic fibrosis - a lethal autosomal recessive disorder. Missense variants that alter a single amino acid in the CFTR protein are among the most common cystic fibrosis variants, yet tools for accurately predicting molecular consequences of missense variants have been limited to date. AlphaMissense (AM) is a new technology that predicts the pathogenicity of missense variants based on dual learned protein structure and evolutionary features. Here, we evaluated the ability of AM to predict the pathogenicity of CFTR missense variants. AM predicted a high pathogenicity for CFTR residues overall, resulting in a high false positive rate and fair classification performance on CF variants from the CFTR2.org database. AM pathogenicity score correlated modestly with pathogenicity metrics from persons with CF including sweat chloride level, pancreatic insufficiency rate, and Pseudomonas aeruginosa infection rate. Correlation was also modest with CFTR trafficking and folding competency in vitro. By contrast, the AM score correlated well with CFTR channel function in vitro - demonstrating the dual structure and evolutionary training approach learns important functional information despite lacking such data during training. Different performance across metrics indicated AM may determine if polymorphisms in CFTR are recessive CF variants yet cannot differentiate mechanistic effects or the nature of pathophysiology. Finally, AM predictions offered limited utility to inform on the pharmacological response of CF variants i.e., theratype. Development of new approaches to differentiate the biochemical and pharmacological properties of CFTR variants is therefore still needed to refine the targeting of emerging precision CF therapeutics.

Elexacaftor/VX-445-mediated CFTR interactome remodeling reveals differential correction driven by mutation-specific translational dynamics.

Cystic fibrosis (CF) is one of the most prevalent lethal genetic diseases with over 2000 identified mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene. Pharmacological chaperones such as lumacaftor (VX-809), tezacaftor (VX-661), and elexacaftor (VX-445) treat mutation-induced defects by stabilizing CFTR and are called correctors. These correctors improve proper folding and thus facilitate processing and trafficking to increase the amount of functional CFTR on the cell surface. Yet, CFTR variants display differential responses to each corrector. Here, we report that variants P67L and L206W respond similarly to VX-809 but divergently to VX-445 with P67L exhibiting little rescue when treated with VX-445. We investigate the underlying cellular mechanisms of how CFTR biogenesis is altered by correctors in these variants. Affinity purification-mass spectrometry multiplexed with isobaric tandem mass tags was used to quantify CFTR protein-protein interaction changes between variants P67L and L206W. VX-445 facilitates unique proteostasis factor interactions especially in translation, folding, and degradation pathways in a CFTR variant-dependent manner. A number of these interacting proteins knocked down by siRNA, such as ribosomal subunit proteins, moderately rescued fully glycosylated P67L. Importantly, these knockdowns sensitize P67L to VX-445 and further enhance the trafficking correction of this variant. Partial inhibition of protein translation also mildly sensitizes P67L CFTR to VX-445 correction, supporting a role for translational dynamics in the rescue mechanism of VX-445. Our results provide a better understanding of VX-445 biological mechanism of action and reveal cellular targets that may sensitize nonresponsive CFTR variants to known and available correctors.

CFTR Folding: From Structure and Proteostasis to Cystic Fibrosis Personalized Medicine.

Cystic fibrosis (CF) is a lethal genetic disease caused by mutations in the chloride ion channel cystic fibrosis transmembrane conductance regulator (CFTR). Class-II mutants of CFTR lack intermolecular interactions important for CFTR structural stability and lead to misfolding. Misfolded CFTR is detected by a diverse suite of proteostasis factors that preferentially bind and route mutant CFTR toward premature degradation, resulting in reduced plasma membrane CFTR levels and impaired chloride ion conductance associated with CF. CF treatment has been vastly improved over the past decade by the availability of small molecules called correctors. Correctors directly bind CFTR, stabilize its structure by conferring thermodynamically favorable interactions that compensate for mutations, and thereby lead to downstream folding fidelity. However, each of over 100 Class-II CF causing mutations causes unique structural defects and shows a unique response to drug treatment, described as theratype. Understanding CFTR structural defects, the proteostasis factors evaluating those defects, and the stabilizing effects of CFTR correctors will illuminate a path toward personalized medicine for CF. Here, we review recent advances in our understanding of CFTR folding, focusing on structure, corrector binding sites, the mechanisms of proteostasis factors that evaluate CFTR, and the implications for CF personalized medicine.

Elexacaftor/VX-445-mediated CFTR interactome remodeling reveals differential correction driven by mutation-specific translational dynamics.

Cystic fibrosis (CF) is one of the most prevalent lethal genetic diseases with over 2000 identified mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene. Pharmacological chaperones such as Lumacaftor (VX-809), Tezacaftor (VX-661) and Elexacaftor (VX-445) treat mutation-induced defects by stabilizing CFTR and are called correctors. These correctors improve proper folding and thus facilitate processing and trafficking to increase the amount of functional CFTR on the cell surface. Yet, CFTR variants display differential responses to each corrector. Here, we report variants P67L and L206W respond similarly to VX-809 but divergently to VX-445 with P67L exhibiting little rescue when treated with VX-445. We investigate the underlying cellular mechanisms of how CFTR biogenesis is altered by correctors in these variants. Affinity purification-mass spectrometry (AP-MS) multiplexed with isobaric Tandem Mass Tags (TMT) was used to quantify CFTR protein-protein interaction changes between variants P67L and L206W. VX-445 facilitates unique proteostasis factor interactions especially in translation, folding, and degradation pathways in a CFTR variant-dependent manner. A number of these interacting proteins knocked down by siRNA, such as ribosomal subunit proteins, moderately rescued fully glycosylated P67L. Importantly, these knock-downs sensitize P67L to VX-445 and further enhance the correction of this variant. Our results provide a better understanding of VX-445 biological mechanism of action and reveal cellular targets that may sensitize unresponsive CFTR variants to known and available correctors.

Benchmarking AlphaFold2 on peptide structure prediction.

Recent advancements in computational tools have allowed protein structure prediction with high accuracy. Computational prediction methods have been used for modeling many soluble and membrane proteins, but the performance of these methods in modeling peptide structures has not yet been systematically investigated. We benchmarked the accuracy of AlphaFold2 in predicting 588 peptide structures between 10 and 40 amino acids using experimentally determined NMR structures as reference. Our results showed AlphaFold2 predicts α-helical, ß-hairpin, and disulfide-rich peptides with high accuracy. AlphaFold2 performed at least as well if not better than alternative methods developed specifically for peptide structure prediction. AlphaFold2 showed several shortcomings in predicting Φ/Ψ angles, disulfide bond patterns, and the lowest RMSD structures failed to correlate with lowest pLDDT ranked structures. In summary, computation can be a powerful tool to predict peptide structures, but additional steps may be necessary to analyze and validate the results.

Distinct proteostasis states drive pharmacologic chaperone susceptibility for cystic fibrosis transmembrane conductance regulator misfolding mutants.

Pharmacological chaperones represent a class of therapeutic compounds for treating protein misfolding diseases. One of the most prominent examples is the FDA-approved pharmacological chaperone lumacaftor (VX-809), which has transformed cystic fibrosis (CF) therapy. CF is a fatal disease caused by mutations in the CF transmembrane conductance regulator (CFTR). VX-809 corrects folding of F508del CFTR, the most common patient mutation, yet F508del exhibits only mild VX-809 response. In contrast, rarer mutations P67L and L206W are hyperresponsive to VX-809, while G85E is nonresponsive. Despite the clinical success of VX-809, the mechanistic origin for the distinct susceptibility of mutants remains unclear. Here we use interactomics to characterize the impact of VX-809 on proteostasis interactions of P67L and L206W and compare these with F508del and G85E. We determine that hyperresponsive mutations P67L and L206W exhibit decreased interactions with proteasomal and autophagy degradation machinery compared with F508del and G85E. We then show inhibiting the proteasome attenuates P67L and L206W VX-809 response. Our data suggest a previously unidentified but required role for protein degradation in VX-809 correction. Furthermore, we present an approach for identifying proteostasis characteristics of mutant-specific therapeutic response to pharmacological chaperones.

Structural Comparative Modeling of Multi-Domain F508del CFTR.

Cystic fibrosis (CF) is a rare genetic disease caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR), an epithelial anion channel expressed in several vital organs. Absence of functional CFTR results in imbalanced osmotic equilibrium and subsequent mucus build up in the lungs-which increases the risk of infection and eventually causes death. CFTR is an ATP-binding cassette (ABC) transporter family protein composed of two transmembrane domains (TMDs), two nucleotide binding domains (NBDs), and an unstructured regulatory domain. The most prevalent patient mutation is the deletion of F508 (F508del), making F508del CFTR the primary target for current FDA approved CF therapies. However, no experimental multi-domain F508del CFTR structure has been determined and few studies have modeled F508del using multi-domain WT CFTR structures. Here, we used cryo-EM density data and Rosetta comparative modeling (RosettaCM) to compare a F508del model with published experimental data on CFTR NBD1 thermodynamics. We then apply this modeling method to generate multi-domain WT and F508del CFTR structural models. These models demonstrate the destabilizing effects of F508del on NBD1 and the NBD1/TMD interface in both the inactive and active conformation of CFTR. Furthermore, we modeled F508del/R1070W and F508del bound to the CFTR corrector VX-809. Our models reveal the stabilizing effects of VX-809 on multi-domain models of F508del CFTR and pave the way for rational design of additional drugs that target F508del CFTR for treatment of CF.