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1
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Lublin L, Senderowitz H. Effects of Point Mutations on the Thermal Stability of the NBD1 Domain of hCFTR. J Chem Inf Model 2025; 65:4531-4553. [PMID: 40271665 DOI: 10.1021/acs.jcim.4c01932] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 04/25/2025]
Abstract
Cystic fibrosis (CF) is an autosomal recessive genetic disease caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel. The first nucleotide-binding domain (NBD1) of the CFTR is considered to be a hotspot for CF-causing mutations, and some of these mutations compromise the domain's thermal stability as well as its interactions with other domains. The mechanisms by which such mutations exert their deleterious effects are important in the basic research of this complex disease as well as for the development of mutation-specific therapies. With this in mind, we studied two class-II, severe, CF-causing mutations, L467P and A559T, known to destabilize the domain by 19.3 and 10.7 °C, respectively, and to lead to a misfolded, nonfunctioning CFTR, by conducting microsecond-long molecular dynamics (MD) simulations at an elevated temperature of 410 K on L467P-NBD1 and A559T-NBD1 constructs. For comparison, similar simulations were also performed on the wild-type (WT) construct and on the 6SS-NBD1 and 2PT/M470V-NBD1 constructs, both bearing sets of stabilizing mutations that stabilize the domain by 17.5 and 8.2 °C, respectively. The resulting trajectories were analyzed using multiple metrics, leading to a good correlation between the experimental ΔTm values and the results of the simulations, as well as multiple experimental observations and results of previous modeling efforts. Specifically, our analyses point to specific regions within NBD1 that are substantially affected by the L467P and A559T mutations and, therefore, may play some role in their pathogenesis. Many of these regions are also known to be important for the proper folding and function of the full-length CFTR. Using time-dependent assignment of DSSP elements, we also found that the two mutants follow different disintegration pathways, that of L467P-NBD1 starting in region 464-471 which resides within the F1-like ATP-binding core subdomain and continues in regions 550-562 and 514-523 within the ABCα subdomain whereas that of A559T-NBD1 simultaneously starting at the 550-562 and 514-523 regions. We propose that the analyses presented in this work may pave the way toward the development of L467P and A559T-specific CF therapies and by extension to other mutation-specific therapies for CF and for other diseases involving mutations in NBDs of other proteins.
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Affiliation(s)
- Lior Lublin
- Department of Chemistry, Bar-Ilan University, Ramat-Gan 5290002, Israel
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2
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Baroni D. Unraveling the Mechanism of Action, Binding Sites, and Therapeutic Advances of CFTR Modulators: A Narrative Review. Curr Issues Mol Biol 2025; 47:119. [PMID: 39996840 PMCID: PMC11854517 DOI: 10.3390/cimb47020119] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/07/2025] [Revised: 02/07/2025] [Accepted: 02/08/2025] [Indexed: 02/26/2025] Open
Abstract
Cystic fibrosis (CF) is a recessive genetic disease caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) protein, a chloride and bicarbonate channel localized on the plasma membrane of epithelial cells. Over the last three decades, high-throughput screening assays have been extensively employed in identifying drugs that target specific defects arising from CFTR mutations. The two main categories of such compounds are potentiators, which enhance CFTR gating by increasing the channel's open probability, and correctors, which improve CFTR protein folding and trafficking to the plasma membrane. In addition to these, other investigational molecules include amplifiers and stabilizers, which enhance the levels and the stability of CFTR on the cell surface, and read-through agents that promote the insertion of correct amino acids at premature termination codons. Currently, four CFTR modulators are clinically approved: the potentiator ivacaftor (VX-770), either as monotherapy or in combination with the correctors lumacaftor (VX-809), tezacaftor (VX-661), and elexacaftor (VX-445). Among these, the triple combination VX-445/VX-661/VX-770 (marketed as Trikafta® in the US and Kaftrio® in Europe) has emerged as the most effective CFTR modulator therapy to date, demonstrating significant clinical benefits in phase III trials for patients with at least one F508del CFTR allele. Despite these advancements, the mechanisms of action and binding sites of these modulators on CFTR have only recently begun to be elucidated. A deeper understanding of these mechanisms could provide essential insights for developing more potent and effective modulators, particularly in combination therapies. This narrative review delves into the mechanism of action, binding sites, and combinatorial effects of approved and investigational CFTR modulators, highlighting ongoing efforts to broaden therapeutic options for individuals with CF.
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Affiliation(s)
- Debora Baroni
- Istituto di Biofisica, Consiglio Nazionale delle Ricerche (CNR), Via De Marini, 6, 16149 Genova, Italy
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3
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Schneider-Futschik EK, Zhu Y, Li D, Habgood MD, Nguyen BN, Pankonien I, Amaral MD, Downie LE, Chinnery HR. The role of CFTR in the eye, and the effect of early highly effective modulator treatment for cystic fibrosis on eye health. Prog Retin Eye Res 2024; 103:101299. [PMID: 39245300 DOI: 10.1016/j.preteyeres.2024.101299] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/27/2024] [Revised: 08/14/2024] [Accepted: 09/05/2024] [Indexed: 09/10/2024]
Abstract
Cystic fibrosis transmembrane conductance regulator (CFTR) is a protein that plays a crucial role in various human organs, including the respiratory and digestive systems. Dysfunctional CFTR is the key variant of the lethal genetic disorder, cystic fibrosis (CF). In the past decade, highly effective CFTR modulator therapies, including elexacaftor-tezacaftor-ivacaftor, have revolutionised CF management by correcting the underlying molecular defect to improve patient outcomes and life expectancy. Despite demonstrating multiorgan efficacy, clinical studies have largely overlooked the potential for ocular disturbances with CFTR modulator therapy, with the exception of a few case studies reporting the presence of crystalline lens pathologies in young children on CFTR modulators, and in breastfed infants born to individuals who were on CFTR modulator treatment during pregnancy. CFTR is present in multiple tissues during embryonic development, including the eye, and its expression can be influenced by genetic and environmental factors. This review summarises the role of CFTR in the eye, and the potential impact of CFTR on eye function and vision later in life. This information provides a framework for understanding the use and possible effects of CFTR-modulating therapeutics in the context of eye health, including the potential to leverage the eye for non-invasive and accessible diagnostic and monitoring capabilities in patients with CF.
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Affiliation(s)
- Elena K Schneider-Futschik
- Department of Biochemistry and Pharmacology, School of Biomedical Sciences, Faculty of Medicine, Dentistry and Health Sciences, The University of Melbourne, Parkville, VIC, 3010, Australia.
| | - Yimin Zhu
- Department of Biochemistry and Pharmacology, School of Biomedical Sciences, Faculty of Medicine, Dentistry and Health Sciences, The University of Melbourne, Parkville, VIC, 3010, Australia
| | - Danni Li
- Department of Biochemistry and Pharmacology, School of Biomedical Sciences, Faculty of Medicine, Dentistry and Health Sciences, The University of Melbourne, Parkville, VIC, 3010, Australia
| | - Mark D Habgood
- Department of Biochemistry and Pharmacology, School of Biomedical Sciences, Faculty of Medicine, Dentistry and Health Sciences, The University of Melbourne, Parkville, VIC, 3010, Australia
| | - Bao N Nguyen
- Department of Optometry and Vision Sciences, The University of Melbourne, Parkville, VIC, 3010, Australia
| | - Ines Pankonien
- BioISI - Biosystems & Integrative Sciences Institute, Faculty of Sciences, University of Lisboa, Campo Grande-C8, 1749-016, Lisboa, Portugal
| | - Margarida D Amaral
- BioISI - Biosystems & Integrative Sciences Institute, Faculty of Sciences, University of Lisboa, Campo Grande-C8, 1749-016, Lisboa, Portugal
| | - Laura E Downie
- Department of Optometry and Vision Sciences, The University of Melbourne, Parkville, VIC, 3010, Australia
| | - Holly R Chinnery
- Department of Optometry and Vision Sciences, The University of Melbourne, Parkville, VIC, 3010, Australia; Lions Eye Institute, Nedlands, Western Australia, 6009, Australia; The University of Western Australia, Crawley, Western Australia, 6009, Australia
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4
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van der Sluijs P, Hoelen H, Schmidt A, Braakman I. The Folding Pathway of ABC Transporter CFTR: Effective and Robust. J Mol Biol 2024; 436:168591. [PMID: 38677493 DOI: 10.1016/j.jmb.2024.168591] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/23/2024] [Revised: 04/16/2024] [Accepted: 04/23/2024] [Indexed: 04/29/2024]
Abstract
De novo protein folding into a native three-dimensional structure is indispensable for biological function, is instructed by its amino acid sequence, and occurs along a vectorial trajectory. The human proteome contains thousands of membrane-spanning proteins, whose biosynthesis begins on endoplasmic reticulum-associated ribosomes. Nearly half of all membrane proteins traverse the membrane more than once, including therapeutically important protein families such as solute carriers, G-protein-coupled receptors, and ABC transporters. These mediate a variety of functions like signal transduction and solute transport and are often of vital importance for cell function and tissue homeostasis. Missense mutations in multispan membrane proteins can lead to misfolding and cause disease; an example is the ABC transporter Cystic Fibrosis Transmembrane Conductance Regulator (CFTR). Even though our understanding of multispan membrane-protein folding still is rather rudimental, the cumulative knowledge of 20 years of basic research on CFTR folding has led to development of drugs that modulate the misfolded protein. This has provided the prospect of a life without CF to the vast majority of patients. In this review we describe our understanding of the folding pathway of CFTR in cells, which is modular and tolerates many defects, making it effective and robust. We address how modulator drugs affect folding and function of CFTR, and distinguish protein stability from its folding process. Since the domain architecture of (mammalian) ABC transporters are highly conserved, we anticipate that the insights we discuss here for folding of CFTR may lay the groundwork for understanding the general rules of ABC-transporter folding.
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Affiliation(s)
- Peter van der Sluijs
- Cellular Protein Chemistry, Bijvoet Centre for Biomolecular Research, Utrecht University, 3584 CH Utrecht, the Netherlands.
| | - Hanneke Hoelen
- Cellular Protein Chemistry, Bijvoet Centre for Biomolecular Research, Utrecht University, 3584 CH Utrecht, the Netherlands; Present address: GenDx, Yalelaan 48, 3584 CM Utrecht, The Netherlands
| | - Andre Schmidt
- Cellular Protein Chemistry, Bijvoet Centre for Biomolecular Research, Utrecht University, 3584 CH Utrecht, the Netherlands; 3D-Pharmxchange, Tilburg, the Netherlands
| | - Ineke Braakman
- Cellular Protein Chemistry, Bijvoet Centre for Biomolecular Research, Utrecht University, 3584 CH Utrecht, the Netherlands
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5
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Yeh HI, Sutcliffe KJ, Sheppard DN, Hwang TC. CFTR Modulators: From Mechanism to Targeted Therapeutics. Handb Exp Pharmacol 2024; 283:219-247. [PMID: 35972584 DOI: 10.1007/164_2022_597] [Citation(s) in RCA: 4] [Impact Index Per Article: 4.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 06/15/2023]
Abstract
People with cystic fibrosis (CF) suffer from a multi-organ disorder caused by loss-of-function variants in the gene encoding the epithelial anion channel cystic fibrosis transmembrane conductance regulator (CFTR). Tremendous progress has been made in both basic and clinical sciences over the past three decades since the identification of the CFTR gene. Over 90% of people with CF now have access to therapies targeting dysfunctional CFTR. This success was made possible by numerous studies in the field that incrementally paved the way for the development of small molecules known as CFTR modulators. The advent of CFTR modulators transformed this life-threatening illness into a treatable disease by directly binding to the CFTR protein and correcting defects induced by pathogenic variants. In this chapter, we trace the trajectory of structural and functional studies that brought CF therapies from bench to bedside, with an emphasis on mechanistic understanding of CFTR modulators.
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Affiliation(s)
- Han-I Yeh
- Department of Pharmacology, National Yang Ming Chiao Tung University, Taipei City, Taiwan
- Department of Medical Pharmacology and Physiology, University of Missouri, Columbia, MO, USA
- Dalton Cardiovascular Research Center, University of Missouri, Columbia, MO, USA
| | - Katy J Sutcliffe
- School of Physiology, Pharmacology and Neuroscience, University of Bristol, Bristol, UK
| | - David N Sheppard
- School of Physiology, Pharmacology and Neuroscience, University of Bristol, Bristol, UK
| | - Tzyh-Chang Hwang
- Department of Pharmacology, National Yang Ming Chiao Tung University, Taipei City, Taiwan.
- Department of Medical Pharmacology and Physiology, University of Missouri, Columbia, MO, USA.
- Dalton Cardiovascular Research Center, University of Missouri, Columbia, MO, USA.
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6
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Baroni D, Scarano N, Ludovico A, Brandas C, Parodi A, Lunaccio D, Fossa P, Moran O, Cichero E, Millo E. In Silico and In Vitro Evaluation of the Mechanism of Action of Three VX809-Based Hybrid Derivatives as Correctors of the F508del CFTR Protein. Pharmaceuticals (Basel) 2023; 16:1702. [PMID: 38139828 PMCID: PMC10748060 DOI: 10.3390/ph16121702] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/11/2023] [Revised: 12/05/2023] [Accepted: 12/06/2023] [Indexed: 12/24/2023] Open
Abstract
Cystic fibrosis (CF), the most common autosomal recessive fatal genetic disease in the Caucasian population, is caused by mutations in the gene encoding the cystic fibrosis transmembrane conductance regulator (CFTR), an anion channel that regulates salt and water transport across a variety of secretory epithelia. Deletion of phenylalanine at position 508, F508del, the most common CF-causing mutation, destabilises the CFTR protein, causing folding and trafficking defects that lead to a dramatic reduction in its functional expression. Small molecules called correctors have been developed to rescue processing-defective F508del CFTR. We have combined in silico and in vitro approaches to investigate the mechanism of action and potential as CFTR correctors of three hybrid derivatives (2a, 7a, and 7m) obtained by merging the amino-arylthiazole core with the benzodioxole carboxamide moiety characterising the corrector lumacaftor. Molecular modelling analyses suggested that the three hybrids interact with a putative region located at the MSD1/NBD1 interface. Biochemical analyses confirmed these results, showing that the three molecules affect the expression and stability of the F508del NBD1. Finally, the YFP assay was used to evaluate the influence of the three hybrid derivatives on F508del CFTR function, assessing that their effect is additive to that of the correctors VX661 and VX445. Our study shows that the development and testing of optimised compounds targeting different structural and functional defects of mutant CFTR is the best strategy to provide more effective correctors that could be used alone or in combination as a valuable therapeutic option to treat an even larger cohort of people affected by CF.
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Affiliation(s)
- Debora Baroni
- Istituto di Biofisica, Consiglio Nazionale delle Ricerche (CNR), Via De Marini, 6, 16149 Genova, Italy; (A.L.); (O.M.)
| | - Naomi Scarano
- Department of Pharmacy, Section of Medicinal Chemistry, School of Medical and Pharmaceutical Sciences, University of Genova, Viale Benedetto XV, 3, 16132 Genoa, Italy; (N.S.); (P.F.)
| | - Alessandra Ludovico
- Istituto di Biofisica, Consiglio Nazionale delle Ricerche (CNR), Via De Marini, 6, 16149 Genova, Italy; (A.L.); (O.M.)
| | - Chiara Brandas
- Istituto di Biofisica, Consiglio Nazionale delle Ricerche (CNR), Via De Marini, 6, 16149 Genova, Italy; (A.L.); (O.M.)
| | - Alice Parodi
- Department of Experimental Medicine, Section of Biochemistry, University of Genoa, Viale Benedetto XV 1, 16132 Genova, Italy; (A.P.); (D.L.); (E.M.)
| | - Dario Lunaccio
- Department of Experimental Medicine, Section of Biochemistry, University of Genoa, Viale Benedetto XV 1, 16132 Genova, Italy; (A.P.); (D.L.); (E.M.)
| | - Paola Fossa
- Department of Pharmacy, Section of Medicinal Chemistry, School of Medical and Pharmaceutical Sciences, University of Genova, Viale Benedetto XV, 3, 16132 Genoa, Italy; (N.S.); (P.F.)
| | - Oscar Moran
- Istituto di Biofisica, Consiglio Nazionale delle Ricerche (CNR), Via De Marini, 6, 16149 Genova, Italy; (A.L.); (O.M.)
| | - Elena Cichero
- Department of Pharmacy, Section of Medicinal Chemistry, School of Medical and Pharmaceutical Sciences, University of Genova, Viale Benedetto XV, 3, 16132 Genoa, Italy; (N.S.); (P.F.)
| | - Enrico Millo
- Department of Experimental Medicine, Section of Biochemistry, University of Genoa, Viale Benedetto XV 1, 16132 Genova, Italy; (A.P.); (D.L.); (E.M.)
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7
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Soya N, Xu H, Roldan A, Yang Z, Ye H, Jiang F, Premchandar A, Veit G, Cole SPC, Kappes J, Hegedüs T, Lukacs GL. Folding correctors can restore CFTR posttranslational folding landscape by allosteric domain-domain coupling. Nat Commun 2023; 14:6868. [PMID: 37891162 PMCID: PMC10611759 DOI: 10.1038/s41467-023-42586-8] [Citation(s) in RCA: 8] [Impact Index Per Article: 4.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/16/2022] [Accepted: 10/16/2023] [Indexed: 10/29/2023] Open
Abstract
The folding/misfolding and pharmacological rescue of multidomain ATP-binding cassette (ABC) C-subfamily transporters, essential for organismal health, remain incompletely understood. The ABCC transporters core consists of two nucleotide binding domains (NBD1,2) and transmembrane domains (TMD1,2). Using molecular dynamic simulations, biochemical and hydrogen deuterium exchange approaches, we show that the mutational uncoupling or stabilization of NBD1-TMD1/2 interfaces can compromise or facilitate the CFTR(ABCC7)-, MRP1(ABCC1)-, and ABCC6-transporters posttranslational coupled domain-folding in the endoplasmic reticulum. Allosteric or orthosteric binding of VX-809 and/or VX-445 folding correctors to TMD1/2 can rescue kinetically trapped CFTR posttranslational folding intermediates of cystic fibrosis (CF) mutants of NBD1 or TMD1 by global rewiring inter-domain allosteric-networks. We propose that dynamic allosteric domain-domain communications not only regulate ABCC-transporters function but are indispensable to tune the folding landscape of their posttranslational intermediates. These allosteric networks can be compromised by CF-mutations, and reinstated by correctors, offering a framework for mechanistic understanding of ABCC-transporters (mis)folding.
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Affiliation(s)
- Naoto Soya
- Department of Physiology and Biochemistry, McGill University, Montréal, QC, Canada
| | - Haijin Xu
- Department of Physiology and Biochemistry, McGill University, Montréal, QC, Canada
| | - Ariel Roldan
- Department of Physiology and Biochemistry, McGill University, Montréal, QC, Canada
| | - Zhengrong Yang
- Heersink School of Medicine, University of Alabama School of Medicine, Birmingham, AL, USA
| | - Haoxin Ye
- Department of Physiology and Biochemistry, McGill University, Montréal, QC, Canada
| | - Fan Jiang
- Heersink School of Medicine, University of Alabama School of Medicine, Birmingham, AL, USA
| | - Aiswarya Premchandar
- Department of Physiology and Biochemistry, McGill University, Montréal, QC, Canada
| | - Guido Veit
- Department of Physiology and Biochemistry, McGill University, Montréal, QC, Canada
| | - Susan P C Cole
- Division of Cancer Biology and Genetics, Department of Pathology and Molecular Medicine, Queen's University Cancer Research Institute, Kingston, ON, Canada
| | - John Kappes
- Heersink School of Medicine, University of Alabama School of Medicine, Birmingham, AL, USA
| | - Tamás Hegedüs
- Department of Biophysics and Radiation Biology, Semmelweis University, 1085, Budapest, Hungary
- ELKH-SE Biophysical Virology Research Group, Eötvös Loránd Research Network, Budapest, Hungary
| | - Gergely L Lukacs
- Department of Physiology and Biochemistry, McGill University, Montréal, QC, Canada.
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8
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Soya N, Xu H, Roldan A, Yang Z, Ye H, Jiang F, Premchandar A, Veit G, Cole SPC, Kappes J, Hegedus T, Lukacs GL. Folding correctors can restore CFTR posttranslational folding landscape by allosteric domain-domain coupling. BIORXIV : THE PREPRINT SERVER FOR BIOLOGY 2023:2023.10.19.563107. [PMID: 37905074 PMCID: PMC10614980 DOI: 10.1101/2023.10.19.563107] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 11/02/2023]
Abstract
The folding/misfolding and pharmacological rescue of multidomain ATP-binding cassette (ABC) C-subfamily transporters, essential for organismal health, remain incompletely understood. The ABCC transporters core consists of two nucleotide binding domains (NBD1,2) and transmembrane domains (TMD1,2). Using molecular dynamic simulations, biochemical and hydrogen deuterium exchange approaches, we show that the mutational uncoupling or stabilization of NBD1-TMD1/2 interfaces can compromise or facilitate the CFTR(ABCC7)-, MRP1(ABCC1)-, and ABCC6-transporters posttranslational coupled domain-folding in the endoplasmic reticulum. Allosteric or orthosteric binding of VX-809 and/or VX-445 folding correctors to TMD1/2 can rescue kinetically trapped CFTR post-translational folding intermediates of cystic fibrosis (CF) mutants of NBD1 or TMD1 by global rewiring inter-domain allosteric-networks. We propose that dynamic allosteric domain-domain communications not only regulate ABCC-transporters function but are indispensable to tune the folding landscape of their post-translational intermediates. These allosteric networks can be compromised by CF-mutations, and reinstated by correctors, offering a framework for mechanistic understanding of ABCC-transporters (mis)folding. One-Sentence Summary Allosteric interdomain communication and its modulation are critical determinants of ABCC-transporters post-translational conformational biogenesis, misfolding, and pharmacological rescue.
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9
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Bongiorno R, Ludovico A, Moran O, Baroni D. Elexacaftor Mediates the Rescue of F508del CFTR Functional Expression Interacting with MSD2. Int J Mol Sci 2023; 24:12838. [PMID: 37629017 PMCID: PMC10454486 DOI: 10.3390/ijms241612838] [Citation(s) in RCA: 2] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/03/2023] [Revised: 08/09/2023] [Accepted: 08/11/2023] [Indexed: 08/27/2023] Open
Abstract
Cystic fibrosis (CF) is one of the most frequent lethal autosomal recessive diseases affecting the Caucasian population. It is caused by loss of function variants of the cystic fibrosis transmembrane conductance regulator (CFTR), a membrane protein located on the apical side of epithelial cells. The most prevalent CF-causing mutation, the deletion of phenylalanine at position 508 (F508del), is characterized by folding and trafficking defects, resulting in the decreased functional expression of the protein on the plasma membrane. Two classes of small-molecule modulators, termed potentiators and correctors, respectively, have been developed to rescue either the gating or the cellular processing of defective F508del CFTR. Kaftrio, a next-generation triple-combination drug, consisting of the potentiator ivacaftor (VX770) and the two correctors tezacaftor (VX661) and elexacaftor (VX445), has been demonstrated to be a life-changing therapeutic modality for the majority of people with CF worldwide. While the mechanism of action of VX770 and VX661 is almost known, the precise mechanism of action and binding site of VX445 have not been conclusively determined. We investigated the activity of VX445 on mutant F508del to identify the protein domains whose expression is mostly affected by this corrector and to disclose its mechanisms of action. Our biochemical analyses revealed that VX445 specifically improves the expression and the maturation of MSD2, heterologously expressed in HEK 293 cells, and confirmed that its effect on the functional expression of defective F508del CFTR is additive either with type I or type II CFTR correctors. We are confident that our study will help to make a step forward in the comprehension of the etiopathology of the CF disease, as well as to give new information for the development and testing of combinations of even more effective correctors able to target mutation-specific defects of the CFTR protein.
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Affiliation(s)
| | | | | | - Debora Baroni
- Istituto di Biofisica, CNR, Via De Marini, 6, 16149 Genova, Italy; (R.B.); (A.L.); (O.M.)
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10
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Li D, Han X, Habgood M, Schneider-Futschik EK. In Utero Mapping and Development Role of CFTR in Lung and Gastrointestinal Tract of Cystic Fibrosis Patients. ACS Pharmacol Transl Sci 2023; 6:355-360. [PMID: 36926454 PMCID: PMC10012249 DOI: 10.1021/acsptsci.2c00233] [Citation(s) in RCA: 4] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/20/2022] [Indexed: 02/16/2023]
Abstract
In cystic fibrosis (CF) the ability of the CF transmembrane conductance regulator (CFTR) protein to mediate chloride and water transport is disrupted. While much progress has been made in CF research leading to effective treatments to improve CFTR function, including small molecule modulators, patients present with varying disease manifestations and responses to therapy. For many CF-affected organs, disease onset is known to occur during in utero development before treatments can be administered and progresses over time leading to irreversible damage to these organs. Thus, the role of functional CFTR protein, in particular, during early development needs to be further elucidated. Studies have detected CFTR proteins at very early gestational stages and revealed temporally and spatially variable CFTR expression patterns in fetuses, suggesting a potential role of CFTR in fetal development. However, the actual mechanisms of how defective CFTR in CF results in fetal morphogenetic abnormalities are yet to be established. This review aims to summarize fetal CFTR expression patterns specifically in the lung, pancreas, and gastrointestinal tract (GIT), as compared to adult patterns. Case studies of structural abnormalities in CF fetuses and newborns and the role of CFTR in fetal development will also be discussed.
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Affiliation(s)
| | | | - Mark Habgood
- Department of Biochemistry
and Pharmacology, School of Biomedical Sciences, Faculty of Medicine,
Dentistry and Health Sciences, The University
of Melbourne, Parkville, VIC 3010, Australia
| | - Elena K. Schneider-Futschik
- Department of Biochemistry
and Pharmacology, School of Biomedical Sciences, Faculty of Medicine,
Dentistry and Health Sciences, The University
of Melbourne, Parkville, VIC 3010, Australia
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11
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Galles GD, Infield DT, Clark CJ, Hemshorn ML, Manikandan S, Fazan F, Rasouli A, Tajkhorshid E, Galpin JD, Cooley RB, Mehl RA, Ahern CA. Tuning phenylalanine fluorination to assess aromatic contributions to protein function and stability in cells. Nat Commun 2023; 14:59. [PMID: 36599844 PMCID: PMC9813137 DOI: 10.1038/s41467-022-35761-w] [Citation(s) in RCA: 18] [Impact Index Per Article: 9.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/13/2022] [Accepted: 12/27/2022] [Indexed: 01/05/2023] Open
Abstract
The aromatic side-chains of phenylalanine, tyrosine, and tryptophan interact with their environments via both hydrophobic and electrostatic interactions. Determining the extent to which these contribute to protein function and stability is not possible with conventional mutagenesis. Serial fluorination of a given aromatic is a validated method in vitro and in silico to specifically alter electrostatic characteristics, but this approach is restricted to a select few experimental systems. Here, we report a group of pyrrolysine-based aminoacyl-tRNA synthetase/tRNA pairs (tRNA/RS pairs) that enable the site-specific encoding of a varied spectrum of fluorinated phenylalanine amino acids in E. coli and mammalian (HEK 293T) cells. By allowing the cross-kingdom expression of proteins bearing these unnatural amino acids at biochemical scale, these tools may potentially enable the study of biological mechanisms which utilize aromatic interactions in structural and cellular contexts.
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Affiliation(s)
- Grace D Galles
- Department of Molecular Physiology and Biophysics, University of Iowa College of Medicine, Iowa City, IA, USA
- The GCE4All Research Center, Department of Biochemistry & Biophysics, Oregon State University, Corvallis, OR, USA
| | - Daniel T Infield
- Department of Molecular Physiology and Biophysics, University of Iowa College of Medicine, Iowa City, IA, USA
| | - Colin J Clark
- Department of Molecular Physiology and Biophysics, University of Iowa College of Medicine, Iowa City, IA, USA
| | - Marcus L Hemshorn
- The GCE4All Research Center, Department of Biochemistry & Biophysics, Oregon State University, Corvallis, OR, USA
| | - Shivani Manikandan
- Department of Molecular Physiology and Biophysics, University of Iowa College of Medicine, Iowa City, IA, USA
| | - Frederico Fazan
- Department of Molecular Physiology and Biophysics, University of Iowa College of Medicine, Iowa City, IA, USA
| | - Ali Rasouli
- Theoretical and Computational Biophysics Group, NIH Center for Macromolecular Modeling and Bioinformatics, Beckman Institute for Advanced Science and Technology, University of Illinois at Urbana-Champaign, Urbana, IL, USA
| | - Emad Tajkhorshid
- Theoretical and Computational Biophysics Group, NIH Center for Macromolecular Modeling and Bioinformatics, Beckman Institute for Advanced Science and Technology, University of Illinois at Urbana-Champaign, Urbana, IL, USA
| | - Jason D Galpin
- Department of Molecular Physiology and Biophysics, University of Iowa College of Medicine, Iowa City, IA, USA
| | - Richard B Cooley
- The GCE4All Research Center, Department of Biochemistry & Biophysics, Oregon State University, Corvallis, OR, USA
| | - Ryan A Mehl
- The GCE4All Research Center, Department of Biochemistry & Biophysics, Oregon State University, Corvallis, OR, USA
| | - Christopher A Ahern
- Department of Molecular Physiology and Biophysics, University of Iowa College of Medicine, Iowa City, IA, USA.
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12
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Im J, Hillenaar T, Yeoh HY, Sahasrabudhe P, Mijnders M, van Willigen M, Hagos A, de Mattos E, van der Sluijs P, Braakman I. ABC-transporter CFTR folds with high fidelity through a modular, stepwise pathway. Cell Mol Life Sci 2023; 80:33. [PMID: 36609925 PMCID: PMC9825563 DOI: 10.1007/s00018-022-04671-x] [Citation(s) in RCA: 14] [Impact Index Per Article: 7.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/20/2022] [Revised: 11/01/2022] [Accepted: 11/28/2022] [Indexed: 01/09/2023]
Abstract
The question how proteins fold is especially pointed for large multi-domain, multi-spanning membrane proteins with complex topologies. We have uncovered the sequence of events that encompass proper folding of the ABC transporter CFTR in live cells by combining kinetic radiolabeling with protease-susceptibility assays. We found that CFTR folds in two clearly distinct stages. The first, co-translational, stage involves folding of the 2 transmembrane domains TMD1 and TMD2, plus one nucleotide-binding domain, NBD1. The second stage is a simultaneous, post-translational increase in protease resistance for both TMDs and NBD2, caused by assembly of these domains onto NBD1. Our assays probe every 2-3 residues (on average) in CFTR. This in-depth analysis at amino-acid level allows detailed analysis of domain folding and importantly also the next level: assembly of the domains into native, folded CFTR. Defects and changes brought about by medicines, chaperones, or mutations also are amenable to analysis. We here show that the well-known disease-causing mutation F508del, which established cystic fibrosis as protein-folding disease, caused co-translational misfolding of NBD1 but not TMD1 nor TMD2 in stage 1, leading to absence of stage-2 folding. Corrector drugs rescued stage 2 without rescuing NBD1. Likewise, the DxD motif in NBD1 that was identified to be required for export of CFTR from the ER we found to be required already upstream of export as CFTR mutated in this motif phenocopies F508del CFTR. The highly modular and stepwise folding process of such a large, complex protein explains the relatively high fidelity and correctability of its folding.
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Affiliation(s)
- Jisu Im
- Cellular Protein Chemistry, Faculty of Science, Bijvoet Centre for Biomolecular Research, Science for Life, Utrecht University, Padualaan 8, 3584 CH Utrecht, The Netherlands
| | - Tamara Hillenaar
- Cellular Protein Chemistry, Faculty of Science, Bijvoet Centre for Biomolecular Research, Science for Life, Utrecht University, Padualaan 8, 3584 CH Utrecht, The Netherlands
| | - Hui Ying Yeoh
- Cellular Protein Chemistry, Faculty of Science, Bijvoet Centre for Biomolecular Research, Science for Life, Utrecht University, Padualaan 8, 3584 CH Utrecht, The Netherlands ,Present Address: Center of Translational Immunology, University Medical Center Utrecht, Utrecht, The Netherlands
| | - Priyanka Sahasrabudhe
- Cellular Protein Chemistry, Faculty of Science, Bijvoet Centre for Biomolecular Research, Science for Life, Utrecht University, Padualaan 8, 3584 CH Utrecht, The Netherlands ,Present Address: Navigo Proteins GmbH, 06120 Halle, Germany
| | - Marjolein Mijnders
- Cellular Protein Chemistry, Faculty of Science, Bijvoet Centre for Biomolecular Research, Science for Life, Utrecht University, Padualaan 8, 3584 CH Utrecht, The Netherlands ,Present Address: Princess Máxima Center for Pediatric Oncology, 3584 CS Utrecht, The Netherlands
| | - Marcel van Willigen
- Cellular Protein Chemistry, Faculty of Science, Bijvoet Centre for Biomolecular Research, Science for Life, Utrecht University, Padualaan 8, 3584 CH Utrecht, The Netherlands ,Present Address: Julius Clinical Ltd, 3703 CD Zeist, The Netherlands
| | - Azib Hagos
- Cellular Protein Chemistry, Faculty of Science, Bijvoet Centre for Biomolecular Research, Science for Life, Utrecht University, Padualaan 8, 3584 CH Utrecht, The Netherlands
| | - Eduardo de Mattos
- Cellular Protein Chemistry, Faculty of Science, Bijvoet Centre for Biomolecular Research, Science for Life, Utrecht University, Padualaan 8, 3584 CH Utrecht, The Netherlands
| | - Peter van der Sluijs
- Cellular Protein Chemistry, Faculty of Science, Bijvoet Centre for Biomolecular Research, Science for Life, Utrecht University, Padualaan 8, 3584 CH Utrecht, The Netherlands
| | - Ineke Braakman
- Cellular Protein Chemistry, Faculty of Science, Bijvoet Centre for Biomolecular Research, Science for Life, Utrecht University, Padualaan 8, 3584 CH Utrecht, The Netherlands
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13
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Recovery of ΔF508-CFTR Function by Citrate. Nutrients 2022; 14:nu14204283. [PMID: 36296967 PMCID: PMC9610893 DOI: 10.3390/nu14204283] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/26/2022] [Revised: 10/09/2022] [Accepted: 10/10/2022] [Indexed: 11/13/2022] Open
Abstract
Treatment of cystic fibrosis relies so far on expensive and sophisticated drugs. A logical approach to rescuing the defective ΔF508-CFTR protein has not yet been published. Therefore, virtual docking of ATP and CFTR activators to the open conformation of the CFTR protein was performed. A new ATP binding site outside of the two known locations was identified. It was located in the cleft between the nucleotide binding domains NBD1 and NBD2 and comprised six basic amino acids in close proximity. Citrate and isocitrate were also bound to this site. Citrate was evaluated for its action on epithelial cells with intact CFTR and defective ΔF508-CFTR. It activated hyaluronan export from human breast carcinoma cells and iodide efflux, and recovered ΔF508-CFTR from premature intracellular degradation. In conclusion, citrate is an activator for ΔF508-CFTR and increases export by defective ΔF508-CFTR into the extracellular matrix of epithelial cells.
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14
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Ludovico A, Moran O, Baroni D. Modulator Combination Improves In Vitro the Microrheological Properties of the Airway Surface Liquid of Cystic Fibrosis Airway Epithelia. Int J Mol Sci 2022; 23:ijms231911396. [PMID: 36232697 PMCID: PMC9569604 DOI: 10.3390/ijms231911396] [Citation(s) in RCA: 4] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/28/2022] [Revised: 09/18/2022] [Accepted: 09/21/2022] [Indexed: 12/03/2022] Open
Abstract
Cystic fibrosis (CF) is a genetic disease caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) protein, a plasma membrane protein expressed on the apical surface of secretory epithelia of the airways. In the airways, defective or absent function of the CFTR protein determines abnormalities of chloride and bicarbonate secretion and, in general, of the transepithelial homeostasis that lead to alterations of airway surface liquid (ASL) composition and properties. The reduction of ASL volume impairs ciliary beating with the consequent accumulation of a sticky mucus. This situation prevents normal mucociliary clearance, favoring the survival and proliferation of bacteria and contributing to the genesis of the CF pulmonary disease. We explored the potential of some CFTR modulators, namely ivacaftor, tezacaftor, elexacaftor and their combination KaftrioTM, capable of partially recovering the basic defects of the CFTR protein, to ameliorate the transepithelial fluid transport and the viscoelastic properties of the mucus when used singly or in combination. Primary human bronchial epithelial cells obtained from CF and non-CF patients were differentiated into a mucociliated epithelia in order to assess the effects of correctors tezacaftor, elexacaftor and their combination with potentiator ivacaftor on the key properties of ASL, such as fluid reabsorption, viscosity, protein content and pH. The treatment of airway epithelia bearing the deletion of a phenylalanine at position 508 (F508del) in the CFTR gene with tezacaftor and elexacaftor significantly improved the pericilial fluid composition, reducing the fluid reabsorption, correcting the ASL pH and reducing the viscosity of the mucus. KaftrioTM was more effective than single modulators in improving all the evaluated parameters, demonstrating once more that this combination recently approved for patients 6 years and older with cystic fibrosis who have at least one F508del mutation in the CFTR gene represents a valuable tool to defeat CF.
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Affiliation(s)
| | | | - Debora Baroni
- Correspondence: ; Tel.: +39-010-647-5559; Fax: +39-010-647-5500
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15
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Molecular mechanisms of Cystic Fibrosis - how mutations lead to misfunction and guide therapy. Biosci Rep 2022; 42:231430. [PMID: 35707985 PMCID: PMC9251585 DOI: 10.1042/bsr20212006] [Citation(s) in RCA: 19] [Impact Index Per Article: 6.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/03/2022] [Revised: 06/03/2022] [Accepted: 06/13/2022] [Indexed: 11/17/2022] Open
Abstract
Cystic fibrosis, the most common autosomal recessive disorder in Caucasians, is caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene, which encodes a cAMP-activated chloride and bicarbonate channel that regulates ion and water transport in secretory epithelia. Although all mutations lead to the lack or reduction in channel function, the mechanisms through which this occurs are diverse – ranging from lack of full-length mRNA, reduced mRNA levels, impaired folding and trafficking, targeting to degradation, decreased gating or conductance, and reduced protein levels to decreased half-life at the plasma membrane. Here, we review the different molecular mechanisms that cause cystic fibrosis and detail how these differences identify theratypes that can inform the use of directed therapies aiming at correcting the basic defect. In summary, we travel through CFTR life cycle from the gene to function, identifying what can go wrong and what can be targeted in terms of the different types of therapeutic approaches.
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16
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Padányi R, Farkas B, Tordai H, Kiss B, Grubmüller H, Soya N, Lukács GL, Kellermayer M, Hegedűs T. Nanomechanics combined with HDX reveals allosteric drug binding sites of CFTR NBD1. Comput Struct Biotechnol J 2022; 20:2587-2599. [PMID: 35685375 PMCID: PMC9160490 DOI: 10.1016/j.csbj.2022.05.036] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/01/2022] [Revised: 05/17/2022] [Accepted: 05/19/2022] [Indexed: 11/29/2022] Open
Abstract
Cystic fibrosis (CF) is a frequent genetic disease in Caucasians that is caused by the deletion of F508 (ΔF508) in the nucleotide binding domain 1 (NBD1) of the CF transmembrane conductance regulator (CFTR). The ΔF508 compromises the folding energetics of the NBD1, as well as the folding of three other CFTR domains. Combination of FDA approved corrector molecules can efficiently but incompletely rescue the ΔF508-CFTR folding and stability defect. Thus, new pharmacophores that would reinstate the wild-type-like conformational stability of the ΔF508-NBD1 would be highly beneficial. The most prominent molecule, 5-bromoindole-3-acetic acid (BIA) that can thermally stabilize the NBD1 has low potency and efficacy. To gain insights into the NBD1 (un)folding dynamics and BIA binding site localization, we combined molecular dynamics (MD) simulations, atomic force spectroscopy (AFM) and hydrogen-deuterium exchange (HDX) experiments. We found that the NBD1 α-subdomain with three adjacent strands from the β-subdomain plays an important role in early folding steps, when crucial non-native interactions are formed via residue F508. Our AFM and HDX experiments showed that BIA associates with this α-core region and increases the resistance of the ΔF508-NBD1 against mechanical unfolding, a phenomenon that could be exploited in future developments of folding correctors.
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Affiliation(s)
- Rita Padányi
- Department of Biophysics and Radiation Biology, Semmelweis University, Budapest, Hungary
| | - Bianka Farkas
- Department of Biophysics and Radiation Biology, Semmelweis University, Budapest, Hungary
- Faculty of Information Technology and Bionics, Pázmány Péter Catholic University, Budapest, Hungary
| | - Hedvig Tordai
- Department of Biophysics and Radiation Biology, Semmelweis University, Budapest, Hungary
| | - Bálint Kiss
- Department of Biophysics and Radiation Biology, Semmelweis University, Budapest, Hungary
| | - Helmut Grubmüller
- Theoretical and Computational Biophysics, Max Planck Institute for Biophysical Chemistry, Göttingen, Germany
| | - Naoto Soya
- Department of Physiology and Biochemistry, McGill University, Montréal, Quebec, Canada
| | - Gergely L. Lukács
- Department of Physiology and Biochemistry, McGill University, Montréal, Quebec, Canada
| | - Miklós Kellermayer
- Department of Biophysics and Radiation Biology, Semmelweis University, Budapest, Hungary
| | - Tamás Hegedűs
- Department of Biophysics and Radiation Biology, Semmelweis University, Budapest, Hungary
- ELKH-SE Molecular Biophysics Research Group, ELKH, Budapest, Hungary
- Corresponding author at: Department of Biophysics and Radiation Biology, Semmelweis University, Budapest, Hungary.
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17
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Prins S, Corradi V, Sheppard DN, Tieleman DP, Vergani P. Can two wrongs make a right? F508del-CFTR ion channel rescue by second-site mutations in its transmembrane domains. J Biol Chem 2022; 298:101615. [PMID: 35065958 PMCID: PMC8861112 DOI: 10.1016/j.jbc.2022.101615] [Citation(s) in RCA: 4] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/01/2021] [Revised: 01/13/2022] [Accepted: 01/17/2022] [Indexed: 11/20/2022] Open
Abstract
Deletion of phenylalanine 508 (F508del) in the cystic fibrosis transmembrane conductance regulator (CFTR) anion channel is the most common cause of cystic fibrosis. The F508 residue is located on nucleotide-binding domain 1 (NBD1) in contact with the cytosolic extensions of the transmembrane helices, in particular intracellular loop 4 (ICL4). To investigate how absence of F508 at this interface impacts the CFTR protein, we carried out a mutagenesis scan of ICL4 by introducing second-site mutations at 11 positions in cis with F508del. Using an image-based fluorescence assay, we measured how each mutation affected membrane proximity and ion-channel function. The scan strongly validated the effectiveness of R1070W at rescuing F508del defects. Molecular dynamics simulations highlighted two features characterizing the ICL4/NBD1 interface of F508del/R1070W-CFTR: flexibility, with frequent transient formation of interdomain hydrogen bonds, and loosely stacked aromatic sidechains (F1068, R1070W, and F1074, mimicking F1068, F508, and F1074 in WT CFTR). F508del-CFTR displayed a distorted aromatic stack, with F1068 displaced toward the space vacated by F508, while in F508del/R1070F-CFTR, which largely retained F508del defects, R1070F could not form hydrogen bonds and the interface was less flexible. Other ICL4 second-site mutations which partially rescued F508del-CFTR included F1068M and F1074M. Methionine side chains allow hydrophobic interactions without the steric rigidity of aromatic rings, possibly conferring flexibility to accommodate the absence of F508 and retain a dynamic interface. These studies highlight how both hydrophobic interactions and conformational flexibility might be important at the ICL4/NBD1 interface, suggesting possible structural underpinnings of F508del-induced dysfunction.
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Affiliation(s)
- Stella Prins
- Department of Neuroscience, Physiology and Pharmacology, University College London, London, UK
| | - Valentina Corradi
- Department of Biological Sciences, Centre for Molecular Simulation, University of Calgary, Calgary, Alberta, Canada
| | - David N Sheppard
- School of Physiology, Pharmacology and Neuroscience, University of Bristol, Bristol, UK
| | - D Peter Tieleman
- Department of Biological Sciences, Centre for Molecular Simulation, University of Calgary, Calgary, Alberta, Canada
| | - Paola Vergani
- Department of Neuroscience, Physiology and Pharmacology, University College London, London, UK.
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18
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Fiedorczuk K, Chen J. Mechanism of CFTR correction by type I folding correctors. Cell 2022; 185:158-168.e11. [PMID: 34995514 DOI: 10.1016/j.cell.2021.12.009] [Citation(s) in RCA: 110] [Impact Index Per Article: 36.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/22/2021] [Revised: 10/02/2021] [Accepted: 12/09/2021] [Indexed: 02/08/2023]
Abstract
Small molecule chaperones have been exploited as therapeutics for the hundreds of diseases caused by protein misfolding. The most successful examples are the CFTR correctors, which transformed cystic fibrosis therapy. These molecules revert folding defects of the ΔF508 mutant and are widely used to treat patients. To investigate the molecular mechanism of their action, we determined cryo-electron microscopy structures of CFTR in complex with the FDA-approved correctors lumacaftor or tezacaftor. Both drugs insert into a hydrophobic pocket in the first transmembrane domain (TMD1), linking together four helices that are thermodynamically unstable. Mutating residues at the binding site rendered ΔF508-CFTR insensitive to lumacaftor and tezacaftor, underscoring the functional significance of the structural discovery. These results support a mechanism in which the correctors stabilize TMD1 at an early stage of biogenesis, prevent its premature degradation, and thereby allosterically rescuing many disease-causing mutations.
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Affiliation(s)
- Karol Fiedorczuk
- Laboratory of Membrane Biology and Biophysics, The Rockefeller University, New York, NY 10065, USA
| | - Jue Chen
- Laboratory of Membrane Biology and Biophysics, The Rockefeller University, New York, NY 10065, USA; Howard Hughes Medical Institute, Chevy Chase, MD 20815, USA.
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19
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Infield DT, Strickland KM, Gaggar A, McCarty NA. The molecular evolution of function in the CFTR chloride channel. J Gen Physiol 2021; 153:212705. [PMID: 34647973 PMCID: PMC8640958 DOI: 10.1085/jgp.202012625] [Citation(s) in RCA: 13] [Impact Index Per Article: 3.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/10/2020] [Revised: 08/11/2021] [Accepted: 09/09/2021] [Indexed: 12/13/2022] Open
Abstract
The ATP-binding cassette (ABC) transporter superfamily includes many proteins of clinical relevance, with genes expressed in all domains of life. Although most members use the energy of ATP binding and hydrolysis to accomplish the active import or export of various substrates across membranes, the cystic fibrosis transmembrane conductance regulator (CFTR) is the only known animal ABC transporter that functions primarily as an ion channel. Defects in CFTR, which is closely related to ABCC subfamily members that bear function as bona fide transporters, underlie the lethal genetic disease cystic fibrosis. This article seeks to integrate structural, functional, and genomic data to begin to answer the critical question of how the function of CFTR evolved to exhibit regulated channel activity. We highlight several examples wherein preexisting features in ABCC transporters were functionally leveraged as is, or altered by molecular evolution, to ultimately support channel function. This includes features that may underlie (1) construction of an anionic channel pore from an anionic substrate transport pathway, (2) establishment and tuning of phosphoregulation, and (3) optimization of channel function by specialized ligand–channel interactions. We also discuss how divergence and conservation may help elucidate the pharmacology of important CFTR modulators.
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Affiliation(s)
- Daniel T Infield
- Department of Molecular Physiology and Biophysics, University of Iowa, Iowa City, IA
| | | | - Amit Gaggar
- Department of Medicine, University of Alabama at Birmingham, Birmingham, AL.,Gregory Fleming James Cystic Fibrosis Research Center, University of Alabama at Birmingham, Birmingham, AL.,Program in Protease and Matrix Biology, University of Alabama at Birmingham, Birmingham, AL.,Birmingham Veterans Administration Medical Center, Birmingham, AL
| | - Nael A McCarty
- Department of Pediatrics, Emory University, Atlanta, GA.,Children's Healthcare of Atlanta Center for Cystic Fibrosis and Airways Disease Research, Emory University, Atlanta, GA
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20
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Yeh HI, Yu YC, Kuo PL, Tsai CK, Huang HT, Hwang TC. Functional stability of CFTR depends on tight binding of ATP at its degenerate ATP-binding site. J Physiol 2021; 599:4625-4642. [PMID: 34411298 DOI: 10.1113/jp281933] [Citation(s) in RCA: 8] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/23/2021] [Accepted: 08/13/2021] [Indexed: 01/28/2023] Open
Abstract
Opening of the cystic fibrosis transmembrane conductance regulator (CFTR) channel is coupled to the motion of its two nucleotide-binding domains: they form a heterodimer sandwiching two functionally distinct ATP-binding sites (sites 1 and 2). While active ATP hydrolysis in site 2 triggers rapid channel closure, the functional role of stable ATP binding in the catalysis-incompetent (or degenerate) site 1, a feature conserved in many other ATP-binding cassette (ABC) transporter proteins, remains elusive. Here, we found that CFTR loses its prompt responsiveness to ATP after the channel is devoid of ATP for tens to hundreds of seconds. Mutants with weakened ATP binding in site 1 and the most prevalent disease-causing mutation, F508del, are more vulnerable to ATP depletion. In contrast, strengthening ligand binding in site 1 with N6 -(2-phenylethyl)-ATP, a high-affinity ATP analogue, or abolishing ATP hydrolysis in site 2 by the mutation D1370N, helps sustain a durable function of the otherwise unstable mutant channels. Thus, tight binding of ATP in the degenerate ATP-binding site is crucial to the functional stability of CFTR. Small molecules targeting site 1 may bear therapeutic potential to overcome the membrane instability of F508del-CFTR. KEY POINTS: During evolution, many ATP-binding cassette transporters - including the cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel, whose dysfunction causes cystic fibrosis (CF) - lose the ability to hydrolyse ATP in one of the two ATP-binding sites. Here we show that tight ATP binding at this degenerate site in CFTR is central for maintaining the stable, robust function of normal CFTR. We also demonstrate that membrane instability of the most common CF-causing mutant, F508del-CFTR, can be rescued by strengthening ATP binding at CFTR's degenerate site. Our data thus explain an evolutionary puzzle and offer a potential therapeutic strategy for CF.
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Affiliation(s)
- Han-I Yeh
- Dalton Cardiovascular Research Center and Department of Medical Pharmacology and Physiology, University of Missouri-Columbia, Columbia, MO, 65211, USA.,Department of Pharmacology, School of Medicine, National Yang Ming Chiao Tung University, Taipei, Taiwan
| | - Ying-Chun Yu
- Dalton Cardiovascular Research Center and Department of Medical Pharmacology and Physiology, University of Missouri-Columbia, Columbia, MO, 65211, USA
| | - Pei-Lun Kuo
- School of Medicine, National Yang Ming Chiao Tung University, Taipei, Taiwan
| | - Chun-Kuang Tsai
- School of Medicine, National Yang Ming Chiao Tung University, Taipei, Taiwan
| | - Hsin-Tuan Huang
- School of Medicine, National Yang Ming Chiao Tung University, Taipei, Taiwan
| | - Tzyh-Chang Hwang
- Dalton Cardiovascular Research Center and Department of Medical Pharmacology and Physiology, University of Missouri-Columbia, Columbia, MO, 65211, USA.,Department of Pharmacology, School of Medicine, National Yang Ming Chiao Tung University, Taipei, Taiwan
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21
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Scholl D, Sigoillot M, Overtus M, Martinez RC, Martens C, Wang Y, Pardon E, Laeremans T, Garcia-Pino A, Steyaert J, Sheppard DN, Hendrix J, Govaerts C. A topological switch in CFTR modulates channel activity and sensitivity to unfolding. Nat Chem Biol 2021; 17:989-997. [PMID: 34341587 DOI: 10.1038/s41589-021-00844-0] [Citation(s) in RCA: 12] [Impact Index Per Article: 3.0] [Reference Citation Analysis] [Abstract] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/17/2020] [Accepted: 06/28/2021] [Indexed: 12/25/2022]
Abstract
The cystic fibrosis transmembrane conductance regulator (CFTR) anion channel is essential to maintain fluid homeostasis in key organs. Functional impairment of CFTR due to mutations in the cftr gene leads to cystic fibrosis. Here, we show that the first nucleotide-binding domain (NBD1) of CFTR can spontaneously adopt an alternate conformation that departs from the canonical NBD fold previously observed. Crystallography reveals that this conformation involves a topological reorganization of NBD1. Single-molecule fluorescence resonance energy transfer microscopy shows that the equilibrium between the conformations is regulated by adenosine triphosphate binding. However, under destabilizing conditions, such as the disease-causing mutation F508del, this conformational flexibility enables unfolding of the β-subdomain. Our data indicate that, in wild-type CFTR, this conformational transition of NBD1 regulates channel function, but, in the presence of the F508del mutation, it allows domain misfolding and subsequent protein degradation. Our work provides a framework to design conformation-specific therapeutics to prevent noxious transitions.
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Affiliation(s)
- Daniel Scholl
- SFMB, Université Libre de Bruxelles, Brussels, Belgium
| | | | - Marie Overtus
- SFMB, Université Libre de Bruxelles, Brussels, Belgium
| | | | - Chloé Martens
- SFMB, Université Libre de Bruxelles, Brussels, Belgium
| | - Yiting Wang
- School of Physiology, Pharmacology and Neuroscience, University of Bristol, Bristol, UK
| | - Els Pardon
- VIB-VUB center for Structural Biology, VIB, Brussels, Belgium.,Structural Biology Brussels, Vrije Universiteit Brussel, Brussels, Belgium
| | - Toon Laeremans
- VIB-VUB center for Structural Biology, VIB, Brussels, Belgium.,Structural Biology Brussels, Vrije Universiteit Brussel, Brussels, Belgium
| | - Abel Garcia-Pino
- Cellular and Molecular Microbiology, Université Libre de Bruxelles, Gosselies, Belgium
| | - Jan Steyaert
- VIB-VUB center for Structural Biology, VIB, Brussels, Belgium.,Structural Biology Brussels, Vrije Universiteit Brussel, Brussels, Belgium
| | - David N Sheppard
- School of Physiology, Pharmacology and Neuroscience, University of Bristol, Bristol, UK
| | - Jelle Hendrix
- Dynamic Bioimaging Lab, Advanced Optical Microscopy Centre and Biomedical Research Institute, Hasselt University, Diepenbeek, Belgium.,Molecular Imaging and Photonics, Chemistry Department, KU Leuven, Leuven, Belgium
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22
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Della Sala A, Prono G, Hirsch E, Ghigo A. Role of Protein Kinase A-Mediated Phosphorylation in CFTR Channel Activity Regulation. Front Physiol 2021; 12:690247. [PMID: 34211404 PMCID: PMC8240754 DOI: 10.3389/fphys.2021.690247] [Citation(s) in RCA: 12] [Impact Index Per Article: 3.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/13/2021] [Accepted: 05/10/2021] [Indexed: 11/17/2022] Open
Abstract
Cystic fibrosis transmembrane conductance regulator (CFTR) is an anion channel expressed on the apical membrane of epithelial cells, where it plays a pivotal role in chloride transport and overall tissue homeostasis. CFTR constitutes a unique member of the ATP-binding cassette transporter superfamily, due to its distinctive cytosolic regulatory (R) domain carrying multiple phosphorylation sites that allow the tight regulation of channel activity and gating. Mutations in the CFTR gene cause cystic fibrosis, the most common lethal autosomal genetic disease in the Caucasian population. In recent years, major efforts have led to the development of CFTR modulators, small molecules targeting the underlying genetic defect of CF and ultimately rescuing the function of the mutant channel. Recent evidence has highlighted that this class of drugs could also impact on the phosphorylation of the R domain of the channel by protein kinase A (PKA), a key regulatory mechanism that is altered in various CFTR mutants. Therefore, the aim of this review is to summarize the current knowledge on the regulation of the CFTR by PKA-mediated phosphorylation and to provide insights into the different factors that modulate this essential CFTR modification. Finally, the discussion will focus on the impact of CF mutations on PKA-mediated CFTR regulation, as well as on how small molecule CFTR regulators and PKA interact to rescue dysfunctional channels.
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Affiliation(s)
- Angela Della Sala
- Molecular Biotechnology Center, Department of Molecular Biotechnology and Health Sciences, University of Turin, Turin, Italy
| | | | - Emilio Hirsch
- Molecular Biotechnology Center, Department of Molecular Biotechnology and Health Sciences, University of Turin, Turin, Italy.,Kither Biotech S.r.l, Turin, Italy
| | - Alessandra Ghigo
- Molecular Biotechnology Center, Department of Molecular Biotechnology and Health Sciences, University of Turin, Turin, Italy.,Kither Biotech S.r.l, Turin, Italy
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23
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Lopes-Pacheco M, Pedemonte N, Veit G. Discovery of CFTR modulators for the treatment of cystic fibrosis. Expert Opin Drug Discov 2021; 16:897-913. [PMID: 33823716 DOI: 10.1080/17460441.2021.1912732] [Citation(s) in RCA: 37] [Impact Index Per Article: 9.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/26/2022]
Abstract
INTRODUCTION Cystic fibrosis (CF) is a life-threatening inherited disease caused by mutations in the gene encoding the CF transmembrane conductance regulator (CFTR) protein, an anion channel expressed at the apical membrane of secretory epithelia. CF leads to multiorgan dysfunction with progressive deterioration of lung function being the major cause of untimely death. Conventional CF therapies target only symptoms and consequences downstream of the primary genetic defect and the current life expectancy and quality of life of these individuals are still very limited. AREA COVERED CFTR modulator drugs are novel-specialized therapies that enhance or even restore functional expression of CFTR mutants and have been approved for clinical use for individuals with specific CF genotypes. This review summarizes classical approaches used for the pre-clinical development of CFTR correctors and potentiators as well as emerging strategies aiming to accelerate modulator development and expand theratyping efforts. EXPERT OPINION Highly effective CFTR modulator drugs are expected to deeply modify the disease course for the majority of individuals with CF. A multitude of experimental approaches have been established to accelerate the development of novel modulators. CF patient-derived specimens are valuable cell models to predict therapeutic effectiveness of existing (and novel) modulators in a precision medicine approach.
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Affiliation(s)
| | | | - Guido Veit
- Department of Physiology, McGill University, Montréal, Canada
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24
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He L, Kennedy AS, Houck S, Aleksandrov A, Quinney NL, Cyr-Scully A, Cholon DM, Gentzsch M, Randell SH, Ren HY, Cyr DM. DNAJB12 and Hsp70 triage arrested intermediates of N1303K-CFTR for endoplasmic reticulum-associated autophagy. Mol Biol Cell 2021; 32:538-553. [PMID: 33534640 PMCID: PMC8101465 DOI: 10.1091/mbc.e20-11-0688] [Citation(s) in RCA: 28] [Impact Index Per Article: 7.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/03/2020] [Revised: 01/19/2021] [Accepted: 01/26/2021] [Indexed: 11/11/2022] Open
Abstract
The transmembrane Hsp40 DNAJB12 and cytosolic Hsp70 cooperate on the endoplasmic reticulum's (ER) cytoplasmic face to facilitate the triage of nascent polytopic membrane proteins for folding versus degradation. N1303K is a common mutation that causes misfolding of the ion channel CFTR, but unlike F508del-CFTR, biogenic and functional defects in N1303K-CFTR are resistant to correction by folding modulators. N1303K is reported to arrest CFTR folding at a late stage after partial assembly of its N-terminal domains. N1303K-CFTR intermediates are clients of JB12-Hsp70 complexes, maintained in a detergent-soluble state, and have a relatively long 3-h half-life. ER-associated degradation (ERAD)-resistant pools of N1303K-CFTR are concentrated in ER tubules that associate with autophagy initiation sites containing WIPI1, FlP200, and LC3. Destabilization of N1303K-CFTR or depletion of JB12 prevents entry of N1303K-CFTR into the membranes of ER-connected phagophores and traffic to autolysosomes. In contrast, the stabilization of intermediates with the modulator VX-809 promotes the association of N1303K-CFTR with autophagy initiation machinery. N1303K-CFTR is excluded from the ER-exit sites, and its passage from the ER to autolysosomes does not require ER-phagy receptors. DNAJB12 operates in biosynthetically active ER microdomains to triage membrane protein intermediates in a conformation-specific manner for secretion versus degradation via ERAD or selective-ER-associated autophagy.
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Affiliation(s)
- Lihua He
- Department of Cell Biology and Physiology and the Cystic Fibrosis/Pulmonary Research and Treatment Center, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599
| | - Andrew S. Kennedy
- Department of Cell Biology and Physiology and the Cystic Fibrosis/Pulmonary Research and Treatment Center, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599
| | - Scott Houck
- Department of Cell Biology and Physiology and the Cystic Fibrosis/Pulmonary Research and Treatment Center, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599
| | - Andrei Aleksandrov
- Department of Cell Biology and Physiology and the Cystic Fibrosis/Pulmonary Research and Treatment Center, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599
| | - Nancy L. Quinney
- Department of Cell Biology and Physiology and the Cystic Fibrosis/Pulmonary Research and Treatment Center, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599
| | - Alexandra Cyr-Scully
- Department of Cell Biology and Physiology and the Cystic Fibrosis/Pulmonary Research and Treatment Center, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599
| | - Deborah M. Cholon
- Department of Cell Biology and Physiology and the Cystic Fibrosis/Pulmonary Research and Treatment Center, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599
| | - Martina Gentzsch
- Department of Cell Biology and Physiology and the Cystic Fibrosis/Pulmonary Research and Treatment Center, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599
| | - Scott H. Randell
- Department of Cell Biology and Physiology and the Cystic Fibrosis/Pulmonary Research and Treatment Center, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599
| | - Hong Yu Ren
- Department of Cell Biology and Physiology and the Cystic Fibrosis/Pulmonary Research and Treatment Center, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599
| | - Douglas M. Cyr
- Department of Cell Biology and Physiology and the Cystic Fibrosis/Pulmonary Research and Treatment Center, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599
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25
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Sabusap CM, Joshi D, Simhaev L, Oliver KE, Senderowitz H, van Willigen M, Braakman I, Rab A, Sorscher EJ, Hong JS. The CFTR P67L variant reveals a key role for N-terminal lasso helices in channel folding, maturation, and pharmacologic rescue. J Biol Chem 2021; 296:100598. [PMID: 33781744 PMCID: PMC8102917 DOI: 10.1016/j.jbc.2021.100598] [Citation(s) in RCA: 20] [Impact Index Per Article: 5.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/21/2021] [Revised: 03/15/2021] [Accepted: 03/25/2021] [Indexed: 12/14/2022] Open
Abstract
Patients with cystic fibrosis (CF) harboring the P67L variant in the cystic fibrosis transmembrane conductance regulator (CFTR) often exhibit a typical CF phenotype, including severe respiratory compromise. This rare mutation (reported in <300 patients worldwide) responds robustly to CFTR correctors, such as lumacaftor and tezacaftor, with rescue in model systems that far exceed what can be achieved for the archetypical CFTR mutant F508del. However, the specific molecular consequences of the P67L mutation are poorly characterized. In this study, we conducted biochemical measurements following low-temperature growth and/or intragenic suppression, which suggest a mechanism underlying P67L that (1) shares key pathogenic features with F508del, including off-pathway (non-native) folding intermediates, (2) is linked to folding stability of nucleotide-binding domains 1 and 2, and (3) demonstrates pharmacologic rescue that requires domains in the carboxyl half of the protein. We also investigated the "lasso" helices 1 and 2, which occur immediately upstream of P67. Based on limited proteolysis, pulse chase, and molecular dynamics analysis of full-length CFTR and a series of deletion constructs, we argue that P67L and other maturational processing (class 2) defects impair the integrity of the lasso motif and confer misfolding of downstream domains. Thus, amino-terminal missense variants elicit a conformational change throughout CFTR that abrogates maturation while providing a robust substrate for pharmacologic repair.
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Affiliation(s)
- Carleen Mae Sabusap
- Department of Pathology, University of Alabama at Birmingham, Birmingham, Alabama, USA
| | - Disha Joshi
- Department of Pediatrics, Emory University School of Medicine, Atlanta, Georgia, USA
| | - Luba Simhaev
- Department of Chemistry, Bar-Ilan University, Ramat-Gan, Israel
| | - Kathryn E Oliver
- Department of Pediatrics, Emory University School of Medicine, Atlanta, Georgia, USA
| | | | - Marcel van Willigen
- Department of Cellular Protein Chemistry, Utrecht University, Utrecht, Netherlands
| | - Ineke Braakman
- Department of Cellular Protein Chemistry, Utrecht University, Utrecht, Netherlands
| | - Andras Rab
- Department of Pediatrics, Emory University School of Medicine, Atlanta, Georgia, USA
| | - Eric J Sorscher
- Department of Pediatrics, Emory University School of Medicine, Atlanta, Georgia, USA.
| | - Jeong S Hong
- Department of Pediatrics, Emory University School of Medicine, Atlanta, Georgia, USA
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26
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Kleizen B, van Willigen M, Mijnders M, Peters F, Grudniewska M, Hillenaar T, Thomas A, Kooijman L, Peters KW, Frizzell R, van der Sluijs P, Braakman I. Co-Translational Folding of the First Transmembrane Domain of ABC-Transporter CFTR is Supported by Assembly with the First Cytosolic Domain. J Mol Biol 2021; 433:166955. [PMID: 33771570 DOI: 10.1016/j.jmb.2021.166955] [Citation(s) in RCA: 28] [Impact Index Per Article: 7.0] [Reference Citation Analysis] [Abstract] [Key Words] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/19/2020] [Revised: 03/16/2021] [Accepted: 03/16/2021] [Indexed: 11/29/2022]
Abstract
ABC transporters transport a wealth of molecules across membranes and consist of transmembrane and cytosolic domains. Their activity cycle involves a tightly regulated and concerted domain choreography. Regulation is driven by the cytosolic domains and function by the transmembrane domains. Folding of these polytopic multidomain proteins to their functional state is a challenge for cells, which is mitigated by co-translational and sequential events. We here reveal the first stages of co-translational domain folding and assembly of CFTR, the ABC transporter defective in the most abundant rare inherited disease cystic fibrosis. We have combined biosynthetic radiolabeling with protease-susceptibility assays and domain-specific antibodies. The most N-terminal domain, TMD1 (transmembrane domain 1), folds both its hydrophobic and soluble helices during translation: the transmembrane helices pack tightly and the cytosolic N- and C-termini assemble with the first cytosolic helical loop ICL1, leaving only ICL2 exposed. This N-C-ICL1 assembly is strengthened by two independent events: (i) assembly of ICL1 with the N-terminal subdomain of the next domain, cytosolic NBD1 (nucleotide-binding domain 1); and (ii) in the presence of corrector drug VX-809, which rescues cell-surface expression of a range of disease-causing CFTR mutants. Both lead to increased shielding of the CFTR N-terminus, and their additivity implies different modes of action. Early assembly of NBD1 and TMD1 is essential for CFTR folding and positions both domains for the required assembly with TMD2. Altogether, we have gained insights into this first, nucleating, VX-809-enhanced domain-assembly event during and immediately after CFTR translation, involving structures conserved in type-I ABC exporters.
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Affiliation(s)
- Bertrand Kleizen
- Cellular Protein Chemistry, Bijvoet Centre for Biomolecular Research, Science for Life, Faculty of Science, Utrecht University, Utrecht, the Netherlands
| | - Marcel van Willigen
- Cellular Protein Chemistry, Bijvoet Centre for Biomolecular Research, Science for Life, Faculty of Science, Utrecht University, Utrecht, the Netherlands; Julius Clinical Ltd, Broederplein 41-43, 3703 CD Zeist, the Netherlands(‡)
| | - Marjolein Mijnders
- Cellular Protein Chemistry, Bijvoet Centre for Biomolecular Research, Science for Life, Faculty of Science, Utrecht University, Utrecht, the Netherlands; Division of Pediatrics, Wilhelmina Children's Hospital, University Medical Center Utrecht, Utrecht, the Netherlands‡
| | - Florence Peters
- Cellular Protein Chemistry, Bijvoet Centre for Biomolecular Research, Science for Life, Faculty of Science, Utrecht University, Utrecht, the Netherlands
| | - Magda Grudniewska
- Cellular Protein Chemistry, Bijvoet Centre for Biomolecular Research, Science for Life, Faculty of Science, Utrecht University, Utrecht, the Netherlands; GenomeScan B.V, Plesmanlaan 1d, 2333 BZ Leiden, the Netherlands‡
| | - Tamara Hillenaar
- Cellular Protein Chemistry, Bijvoet Centre for Biomolecular Research, Science for Life, Faculty of Science, Utrecht University, Utrecht, the Netherlands
| | - Ann Thomas
- Cellular Protein Chemistry, Bijvoet Centre for Biomolecular Research, Science for Life, Faculty of Science, Utrecht University, Utrecht, the Netherlands; UniQure, Paasheuvelweg 25a, 1105 BP Amsterdam, the Netherlands‡
| | - Laurens Kooijman
- Cellular Protein Chemistry, Bijvoet Centre for Biomolecular Research, Science for Life, Faculty of Science, Utrecht University, Utrecht, the Netherlands; Department of Chemistry, University of Zurich, Winterthurerstrasse 190, 8057 Zürich, Switzerland‡
| | - Kathryn W Peters
- Departments of Pediatrics and Cell Biology, School of Medicine, University of Pittsburgh, Pittsburgh, PA 15261, USA
| | - Raymond Frizzell
- Departments of Pediatrics and Cell Biology, School of Medicine, University of Pittsburgh, Pittsburgh, PA 15261, USA
| | - Peter van der Sluijs
- Cellular Protein Chemistry, Bijvoet Centre for Biomolecular Research, Science for Life, Faculty of Science, Utrecht University, Utrecht, the Netherlands
| | - Ineke Braakman
- Cellular Protein Chemistry, Bijvoet Centre for Biomolecular Research, Science for Life, Faculty of Science, Utrecht University, Utrecht, the Netherlands.
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27
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Discovery of novel VX-809 hybrid derivatives as F508del-CFTR correctors by molecular modeling, chemical synthesis and biological assays. Eur J Med Chem 2020; 208:112833. [DOI: 10.1016/j.ejmech.2020.112833] [Citation(s) in RCA: 6] [Impact Index Per Article: 1.2] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/16/2020] [Revised: 09/04/2020] [Accepted: 09/05/2020] [Indexed: 11/21/2022]
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28
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Characterization of the mechanism of action of RDR01752, a novel corrector of F508del-CFTR. Biochem Pharmacol 2020; 180:114133. [DOI: 10.1016/j.bcp.2020.114133] [Citation(s) in RCA: 9] [Impact Index Per Article: 1.8] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/22/2020] [Revised: 06/30/2020] [Accepted: 06/30/2020] [Indexed: 12/16/2022]
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29
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Wang W, Fu L, Liu Z, Wen H, Rab A, Hong JS, Kirk KL, Rowe SM. G551D mutation impairs PKA-dependent activation of CFTR channel that can be restored by novel GOF mutations. Am J Physiol Lung Cell Mol Physiol 2020; 319:L770-L785. [PMID: 32877225 DOI: 10.1152/ajplung.00262.2019] [Citation(s) in RCA: 7] [Impact Index Per Article: 1.4] [Reference Citation Analysis] [Abstract] [Key Words] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/07/2023] Open
Abstract
G551D is a major disease-associated gating mutation in the cystic fibrosis transmembrane conductance regulator (CFTR) protein, an ATP- and phosphorylation-dependent chloride channel. G551D causes severe cystic fibrosis (CF) disease by disrupting ATP-dependent channel opening; however, whether G551D affects phosphorylation-dependent channel activation is unclear. Here, we use macropatch recording and Ussing chamber approaches to demonstrate that G551D impacts on phosphorylation-dependent activation of CFTR, and PKA-mediated phosphorylation regulates the interaction between the x-loop in nucleotide-binding domain 2 (NBD2) and cytosolic loop (CL) 1. We show that G551D not only disrupts ATP-dependent channel opening but also impairs phosphorylation-dependent channel activation by largely reducing PKA sensitivity consistent with the reciprocal relationship between channel opening/gating, ligand binding, and phosphorylation. Furthermore, we identified two novel GOF mutations: D1341R in the x-loop near the ATP-binding cassette signature motif in NBD2 and D173R in CL1, each of which strongly increased PKA sensitivity both in the wild-type (WT) background and when introduced into G551D-CFTR. When D1341R was combined with a second GOF mutation (e.g., K978C in CL3), we find that the double GOF mutation maximally increased G551D channel activity such that VX-770 had no further effect. We further show that a double charge-reversal mutation of D1341R/D173R-CFTR exhibited similar PKA sensitivity when compared with WT-CFTR. Together, our results suggest that charge repulsion between D173 and D1341 of WT-CFTR normally inhibits channel activation at low PKA activity by reducing PKA sensitivity, and negative allostery by the G551D is coupled to reduced PKA sensitivity of CFTR that can be restored by second GOF mutations.
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Affiliation(s)
- Wei Wang
- Department of Cell, Developmental, and Integrative Biology, University of Alabama at Birmingham, Birmingham, Alabama.,Gregory Fleming James Cystic Fibrosis Research Center, University of Alabama at Birmingham, Birmingham, Alabama
| | - Lianwu Fu
- Department of Cell, Developmental, and Integrative Biology, University of Alabama at Birmingham, Birmingham, Alabama.,Gregory Fleming James Cystic Fibrosis Research Center, University of Alabama at Birmingham, Birmingham, Alabama
| | - Zhiyong Liu
- Gregory Fleming James Cystic Fibrosis Research Center, University of Alabama at Birmingham, Birmingham, Alabama
| | - Hui Wen
- Gregory Fleming James Cystic Fibrosis Research Center, University of Alabama at Birmingham, Birmingham, Alabama
| | - Andras Rab
- Department of Pediatrics, Emory University, Atlanta, Georgia
| | - Jeong S Hong
- Department of Pediatrics, Emory University, Atlanta, Georgia
| | - Kevin L Kirk
- Department of Cell, Developmental, and Integrative Biology, University of Alabama at Birmingham, Birmingham, Alabama.,Gregory Fleming James Cystic Fibrosis Research Center, University of Alabama at Birmingham, Birmingham, Alabama
| | - Steven M Rowe
- Department of Cell, Developmental, and Integrative Biology, University of Alabama at Birmingham, Birmingham, Alabama.,Gregory Fleming James Cystic Fibrosis Research Center, University of Alabama at Birmingham, Birmingham, Alabama.,School of Medicine, University of Alabama at Birmingham, Birmingham, Alabama.,Department of Pediatrics, University of Alabama at Birmingham, Birmingham, Alabama
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30
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Luo S, Feng J, Zhang Y, Yang X, Ma G, Hu T, Xi Y, Tu X, Wang C, Zhang H, Zou Z, Zhang Y. Mutation analysis of the cystic fibrosis transmembrane conductance regulator gene in Chinese congenital absence of vas deferens patients. Gene 2020; 765:145045. [PMID: 32777524 DOI: 10.1016/j.gene.2020.145045] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/15/2020] [Revised: 08/05/2020] [Accepted: 08/06/2020] [Indexed: 10/23/2022]
Abstract
To find the variant spectrum of the cystic fibrosis transmembrane conductance regulator (CFTR) gene, and evaluate its frequent variants in Chinese congenital absence of vas deferens (CAVD) patients. A total of 276 patients with azoospermia and CAVD (aged from 21 to 44 years old) were investigated from May 2013 to September 2019 in the Third Affiliated Hospital of Sun Yat-sen University. Additionally, 50 healthy, unrelated volunteers were recruited as controls (aged from 21 to 46 years old). The 5'-UTR, exons and their flanking side of the CFTR gene were sequenced by high-throughput sequencing technology. The results were compared with those retrieved from the Ensembl Genome Browser. In addition, all 13 novel variants were further confirmed independently by Sanger sequencing and evaluated in the bioinformatics web servers. A schematic of the variant spectrum of the CFTR gene, including 13 novel variants (12 in CAVD patients, one in the control group), is shown, and the frequent variants in Chinese CAVD patients were 5 T (27.54%), c.-8G > C (7.25%), p.Q1352H (5.98%), and p.I556V (3.08%). 5 T was found to be the most frequent variant. p.Q1352H had a significantly high allelic frequency in CAVD patients (P < 0.05). c.-8G > C and p.I556V had high allelic frequencies but showed no difference between patients and controls (P > 0.05). p.Q1352H is the most common and important missense variant in Chinese patients with CAVD, while the pathological effects of C.-8G > C and p.I556V may be weak after evaluation.
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Affiliation(s)
- Shaoge Luo
- Department of Infertility and Sexual Medicine, The Third Affiliated Hospital, Sun Yat-sen University, No. 600, Tianhe Road, Guangzhou 510630, China
| | - Jiarong Feng
- Department of Infertility and Sexual Medicine, The Third Affiliated Hospital, Sun Yat-sen University, No. 600, Tianhe Road, Guangzhou 510630, China
| | - Yanan Zhang
- Department of Infertility and Sexual Medicine, The Third Affiliated Hospital, Sun Yat-sen University, No. 600, Tianhe Road, Guangzhou 510630, China
| | - Xiaojian Yang
- Department of Infertility and Sexual Medicine, The Third Affiliated Hospital, Sun Yat-sen University, No. 600, Tianhe Road, Guangzhou 510630, China
| | - Gongchao Ma
- Department of Infertility and Sexual Medicine, The Third Affiliated Hospital, Sun Yat-sen University, No. 600, Tianhe Road, Guangzhou 510630, China
| | - Tengfei Hu
- Department of Infertility and Sexual Medicine, The Third Affiliated Hospital, Sun Yat-sen University, No. 600, Tianhe Road, Guangzhou 510630, China
| | - Yu Xi
- Department of Infertility and Sexual Medicine, The Third Affiliated Hospital, Sun Yat-sen University, No. 600, Tianhe Road, Guangzhou 510630, China
| | - Xuchong Tu
- Department of Infertility and Sexual Medicine, The Third Affiliated Hospital, Sun Yat-sen University, No. 600, Tianhe Road, Guangzhou 510630, China
| | - Chunlin Wang
- Department of Andrology, Ruikang Hospital Affiliated to Guangxi University of Traditional Chinese Medicine, No.10, Huadong Road, Nanning 530011, China
| | - Hui Zhang
- Department of Infertility and Sexual Medicine, The Third Affiliated Hospital, Sun Yat-sen University, No. 600, Tianhe Road, Guangzhou 510630, China
| | - Zijun Zou
- Department of Infertility and Sexual Medicine, The Third Affiliated Hospital, Sun Yat-sen University, No. 600, Tianhe Road, Guangzhou 510630, China
| | - Yan Zhang
- Department of Infertility and Sexual Medicine, The Third Affiliated Hospital, Sun Yat-sen University, No. 600, Tianhe Road, Guangzhou 510630, China.
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31
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Long QT Syndrome Type 2: Emerging Strategies for Correcting Class 2 KCNH2 ( hERG) Mutations and Identifying New Patients. Biomolecules 2020. [PMID: 32759882 DOI: 10.3390/biom10081144s] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [Abstract] [Key Words] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/25/2023] Open
Abstract
Significant advances in our understanding of the molecular mechanisms that cause congenital long QT syndrome (LQTS) have been made. A wide variety of experimental approaches, including heterologous expression of mutant ion channel proteins and the use of inducible pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) from LQTS patients offer insights into etiology and new therapeutic strategies. This review briefly discusses the major molecular mechanisms underlying LQTS type 2 (LQT2), which is caused by loss-of-function (LOF) mutations in the KCNH2 gene (also known as the human ether-à-go-go-related gene or hERG). Almost half of suspected LQT2-causing mutations are missense mutations, and functional studies suggest that about 90% of these mutations disrupt the intracellular transport, or trafficking, of the KCNH2-encoded Kv11.1 channel protein to the cell surface membrane. In this review, we discuss emerging strategies that improve the trafficking and functional expression of trafficking-deficient LQT2 Kv11.1 channel proteins to the cell surface membrane and how new insights into the structure of the Kv11.1 channel protein will lead to computational approaches that identify which KCNH2 missense variants confer a high-risk for LQT2.
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32
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Ono M, Burgess DE, Schroder EA, Elayi CS, Anderson CL, January CT, Sun B, Immadisetty K, Kekenes-Huskey PM, Delisle BP. Long QT Syndrome Type 2: Emerging Strategies for Correcting Class 2 KCNH2 ( hERG) Mutations and Identifying New Patients. Biomolecules 2020; 10:E1144. [PMID: 32759882 PMCID: PMC7464307 DOI: 10.3390/biom10081144] [Citation(s) in RCA: 25] [Impact Index Per Article: 5.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/17/2020] [Revised: 07/25/2020] [Accepted: 07/27/2020] [Indexed: 12/15/2022] Open
Abstract
Significant advances in our understanding of the molecular mechanisms that cause congenital long QT syndrome (LQTS) have been made. A wide variety of experimental approaches, including heterologous expression of mutant ion channel proteins and the use of inducible pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) from LQTS patients offer insights into etiology and new therapeutic strategies. This review briefly discusses the major molecular mechanisms underlying LQTS type 2 (LQT2), which is caused by loss-of-function (LOF) mutations in the KCNH2 gene (also known as the human ether-à-go-go-related gene or hERG). Almost half of suspected LQT2-causing mutations are missense mutations, and functional studies suggest that about 90% of these mutations disrupt the intracellular transport, or trafficking, of the KCNH2-encoded Kv11.1 channel protein to the cell surface membrane. In this review, we discuss emerging strategies that improve the trafficking and functional expression of trafficking-deficient LQT2 Kv11.1 channel proteins to the cell surface membrane and how new insights into the structure of the Kv11.1 channel protein will lead to computational approaches that identify which KCNH2 missense variants confer a high-risk for LQT2.
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Affiliation(s)
- Makoto Ono
- Department of Physiology, Cardiovascular Research Center, Center for Muscle Biology, University of Kentucky, Lexington, KY 40536, USA; (M.O.); (D.E.B.); (E.A.S.)
| | - Don E. Burgess
- Department of Physiology, Cardiovascular Research Center, Center for Muscle Biology, University of Kentucky, Lexington, KY 40536, USA; (M.O.); (D.E.B.); (E.A.S.)
| | - Elizabeth A. Schroder
- Department of Physiology, Cardiovascular Research Center, Center for Muscle Biology, University of Kentucky, Lexington, KY 40536, USA; (M.O.); (D.E.B.); (E.A.S.)
| | | | - Corey L. Anderson
- Cellular and Molecular Arrhythmia Research Program, University of Wisconsin, Madison, WI 53706, USA; (C.L.A.); (C.T.J.)
| | - Craig T. January
- Cellular and Molecular Arrhythmia Research Program, University of Wisconsin, Madison, WI 53706, USA; (C.L.A.); (C.T.J.)
| | - Bin Sun
- Department of Cellular & Molecular Physiology, Loyola University Chicago, Chicago, IL 60153, USA; (B.S.); (K.I.); (P.M.K.-H.)
| | - Kalyan Immadisetty
- Department of Cellular & Molecular Physiology, Loyola University Chicago, Chicago, IL 60153, USA; (B.S.); (K.I.); (P.M.K.-H.)
| | - Peter M. Kekenes-Huskey
- Department of Cellular & Molecular Physiology, Loyola University Chicago, Chicago, IL 60153, USA; (B.S.); (K.I.); (P.M.K.-H.)
| | - Brian P. Delisle
- Department of Physiology, Cardiovascular Research Center, Center for Muscle Biology, University of Kentucky, Lexington, KY 40536, USA; (M.O.); (D.E.B.); (E.A.S.)
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33
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Uliyakina I, Botelho HM, da Paula AC, Afonso S, Lobo MJ, Felício V, Farinha CM, Amaral MD. Full Rescue of F508del-CFTR Processing and Function by CFTR Modulators Can Be Achieved by Removal of Two Regulatory Regions. Int J Mol Sci 2020; 21:ijms21124524. [PMID: 32630527 PMCID: PMC7350234 DOI: 10.3390/ijms21124524] [Citation(s) in RCA: 8] [Impact Index Per Article: 1.6] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/22/2020] [Revised: 06/20/2020] [Accepted: 06/22/2020] [Indexed: 01/07/2023] Open
Abstract
Cystic Fibrosis (CF) is caused by mutations in the CF Transmembrane conductance Regulator (CFTR), the only ATP-binding cassette (ABC) transporter functioning as a channel. Unique to CFTR is a regulatory domain which includes a highly conformationally dynamic region—the regulatory extension (RE). The first nucleotide-binding domain of CFTR contains another dynamic region—regulatory insertion (RI). Removal of RI rescues the trafficking defect of CFTR with F508del, the most common CF-causing mutation. Here we aimed to assess the impact of RE removal (with/without RI or genetic revertants) on F508del-CFTR trafficking and how CFTR modulator drugs VX-809/lumacaftor and VX-770/ivacaftor rescue these variants. We generated cell lines expressing ΔRE and ΔRI CFTR (with/without genetic revertants) and assessed CFTR expression, stability, plasma membrane levels, and channel activity. Our data demonstrated that ΔRI significantly enhanced rescue of F508del-CFTR by VX-809. While the presence of the RI seems to be precluding full rescue of F508del-CFTR processing by VX-809, this region appears essential to rescue its function by VX-770, suggesting some contradictory role in rescue of F508del-CFTR by these two modulators. This negative impact of RI removal on VX-770-stimulated currents on F508del-CFTR can be compensated by deletion of the RE which also leads to the stabilization of this mutant. Despite both regions being conformationally dynamic, RI precludes F508del-CFTR processing while RE affects mostly its stability and channel opening.
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Patel SD, Bono TR, Rowe SM, Solomon GM. CFTR targeted therapies: recent advances in cystic fibrosis and possibilities in other diseases of the airways. Eur Respir Rev 2020; 29:29/156/190068. [PMID: 32554756 PMCID: PMC9131734 DOI: 10.1183/16000617.0068-2019] [Citation(s) in RCA: 20] [Impact Index Per Article: 4.0] [Reference Citation Analysis] [Abstract] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/13/2019] [Accepted: 03/09/2020] [Indexed: 12/11/2022] Open
Abstract
Cystic fibrosis transmembrane conductance regulator (CFTR) is an ion transporter that regulates mucus hydration, viscosity and acidity of the airway epithelial surface. Genetic defects in CFTR impair regulation of mucus homeostasis, causing severe defects of mucociliary clearance as seen in cystic fibrosis. Recent work has established that CFTR dysfunction can be acquired in chronic obstructive pulmonary disease (COPD) and may also contribute to other diseases that share clinical features of cystic fibrosis, such as asthma, allergic bronchopulmonary aspergillosis and bronchiectasis. Protean causes of CFTR dysfunction have been identified including cigarette smoke exposure, toxic metals and downstream effects of neutrophil activation pathways. Recently, CFTR modulators, small molecule agents that potentiate CFTR or restore diminished protein levels at the cell surface, have been successfully developed for various CFTR gene defects, prompting interest in their use to treat diseases of acquired dysfunction. The spectrum of CFTR dysfunction, strategies for CFTR modulation, and candidate diseases for CFTR modulation beyond cystic fibrosis will be reviewed in this manuscript. CFTR dysfunction may be part of the pathophysiology of many diseases of the airways. Exploration of mechanisms of dysfunction and options for CFTR-directed therapies are examined in this article. http://bit.ly/33o6nDu
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Affiliation(s)
- Sheylan D Patel
- Dept of Medicine, University of Alabama at Birmingham, Birmingham, AL, USA.,Both authors contributed equally
| | - Taylor R Bono
- Dept of Medicine, University of Alabama at Birmingham, Birmingham, AL, USA.,The Gregory Fleming James Cystic Fibrosis Research Center, University of Alabama at Birmingham, Birmingham, AL, USA.,Both authors contributed equally
| | - Steven M Rowe
- Dept of Medicine, University of Alabama at Birmingham, Birmingham, AL, USA .,The Gregory Fleming James Cystic Fibrosis Research Center, University of Alabama at Birmingham, Birmingham, AL, USA
| | - George M Solomon
- Dept of Medicine, University of Alabama at Birmingham, Birmingham, AL, USA.,The Gregory Fleming James Cystic Fibrosis Research Center, University of Alabama at Birmingham, Birmingham, AL, USA
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Dokholyan NV. Experimentally-driven protein structure modeling. J Proteomics 2020; 220:103777. [PMID: 32268219 PMCID: PMC7214187 DOI: 10.1016/j.jprot.2020.103777] [Citation(s) in RCA: 20] [Impact Index Per Article: 4.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/18/2019] [Revised: 03/17/2020] [Accepted: 04/02/2020] [Indexed: 11/25/2022]
Abstract
Revolutions in natural and exact sciences started at the dawn of last century have led to the explosion of theoretical, experimental, and computational approaches to determine structures of molecules, complexes, as well as their rich conformational dynamics. Since different experimental methods produce information that is attributed to specific time and length scales, corresponding computational methods have to be tailored to these scales and experiments. These methods can be then combined and integrated in scales, hence producing a fuller picture of molecular structure and motion from the "puzzle pieces" offered by various experiments. Here, we describe a number of computational approaches to utilize experimental data to glance into structure of proteins and understand their dynamics. We will also discuss the limitations and the resolution of the constraints-based modeling approaches. SIGNIFICANCE: Experimentally-driven computational structure modeling and determination is a rapidly evolving alternative to traditional approaches for molecular structure determination. These new hybrid experimental-computational approaches are proving to be a powerful microscope to glance into the structural features of intrinsically or partially disordered proteins, dynamics of molecules and complexes. In this review, we describe various approaches in the field of experimentally-driven computational structure modeling.
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Affiliation(s)
- Nikolay V Dokholyan
- Department of Pharmacology, Penn State University College of Medicine, Hershey, PA 17033, USA; Department of Biochemistry & Molecular Biology, Penn State College of Medicine, Hershey, PA 17033, USA.; Department of Chemistry, Pennsylvania State University, University Park, PA 16802, USA.; Department of Biomedical Engineering, Pennsylvania State University, University Park, PA 16802, USA.
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Rang C, Keating D, Wilson J, Kotsimbos T. Re-imagining cystic fibrosis care: next generation thinking. Eur Respir J 2020; 55:13993003.02443-2019. [PMID: 32139465 DOI: 10.1183/13993003.02443-2019] [Citation(s) in RCA: 7] [Impact Index Per Article: 1.4] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/20/2019] [Accepted: 02/20/2020] [Indexed: 12/26/2022]
Abstract
Cystic fibrosis (CF) is a common multi-system genetically inherited condition, predominately found in individuals of Caucasian decent. Since the identification of the cystic fibrosis (CF) transmembrane conductance regulator (CFTR) gene in 1989, and the subsequent improvement in understanding of CF pathophysiology, significant increases in life-expectancy have followed. Initially this was related to improvements in the management and systems of care for treating the various affected organ systems. These cornerstone treatments are still essential for CF patients born today. However, over the last decade, the major advance has been in therapies that target the resultant genetic defect: the dysfunctional CFTR protein. Small molecule agents that target this dysfunctional protein via a variety of mechanisms have led to lung function improvements, reductions in pulmonary exacerbation rates and increases in weight and quality-of-life indices. As more patients receive these agents earlier and earlier in life, it is likely that general CF care will increasingly pivot around these specific therapies, although it is also likely that effects other than those identified in the initial trials will be discovered and need to be managed. Despite great excitement for modulator therapies, they are unlikely to be suitable or available for all; whether this is due to a lack of availability for specific CFTR mutations, drug-reactions or the health economic set-up in certain countries. Nevertheless, the CF community must be applauded for its ongoing focus on research and development for this life-limiting disease. With time, personalised individualised therapy would ideally be the mainstay of CF care.
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Affiliation(s)
- Catherine Rang
- Cystic Fibrosis Service, Dept of Respiratory Medicine, Alfred Health, Melbourne, Australia
| | - Dominic Keating
- Cystic Fibrosis Service, Dept of Respiratory Medicine, Alfred Health, Melbourne, Australia.,Dept of Medicine, Monash University, Alfred Campus, Melbourne, Australia
| | - John Wilson
- Cystic Fibrosis Service, Dept of Respiratory Medicine, Alfred Health, Melbourne, Australia.,Dept of Medicine, Monash University, Alfred Campus, Melbourne, Australia
| | - Tom Kotsimbos
- Cystic Fibrosis Service, Dept of Respiratory Medicine, Alfred Health, Melbourne, Australia.,Dept of Medicine, Monash University, Alfred Campus, Melbourne, Australia
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37
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Recent Strategic Advances in CFTR Drug Discovery: An Overview. Int J Mol Sci 2020; 21:ijms21072407. [PMID: 32244346 PMCID: PMC7177952 DOI: 10.3390/ijms21072407] [Citation(s) in RCA: 5] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/10/2020] [Revised: 03/25/2020] [Accepted: 03/27/2020] [Indexed: 12/13/2022] Open
Abstract
Cystic fibrosis transmembrane conductance regulator (CFTR)-rescuing drugs have already transformed cystic fibrosis (CF) from a fatal disease to a treatable chronic condition. However, new-generation drugs able to bind CFTR with higher specificity/affinity and to exert stronger therapeutic benefits and fewer side effects are still awaited. Computational methods and biosensors have become indispensable tools in the process of drug discovery for many important human pathologies. Instead, they have been used only piecemeal in CF so far, calling for their appropriate integration with well-tried CF biochemical and cell-based models to speed up the discovery of new CFTR-rescuing drugs. This review will give an overview of the available structures and computational models of CFTR and of the biosensors, biochemical and cell-based assays already used in CF-oriented studies. It will also give the reader some insights about how to integrate these tools as to improve the efficiency of the drug discovery process targeted to CFTR.
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Baaklini I, Gonçalves CDC, Lukacs GL, Young JC. Selective Binding of HSC70 and its Co-Chaperones to Structural Hotspots on CFTR. Sci Rep 2020; 10:4176. [PMID: 32144307 PMCID: PMC7060200 DOI: 10.1038/s41598-020-61107-x] [Citation(s) in RCA: 19] [Impact Index Per Article: 3.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/04/2019] [Accepted: 02/07/2020] [Indexed: 12/17/2022] Open
Abstract
Mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) channel cause cystic fibrosis. Chaperones, including HSC70, DNAJA1 and DNAJA2, play key roles in both the folding and degradation of wild-type and mutant CFTR at multiple cellular locations. DNAJA1 and HSC70 promote the folding of newly synthesized CFTR at the endoplasmic reticulum (ER), but are required for the rapid turnover of misfolded channel at the plasma membrane (PM). DNAJA2 and HSC70 are also involved in the ER-associated degradation (ERAD) of misfolded CFTR, while they assist the refolding of destabilized channel at the PM. These outcomes may depend on the binding of chaperones to specific sites within CFTR, which would be exposed in non-native states. A CFTR peptide library was used to identify binding sites for HSC70, DNAJA1 and DNAJA2, validated by competition and functional assays. Each chaperone had a distinct binding pattern, and sites were distributed between the surfaces of the CFTR cytosolic domains, and domain interfaces known to be important for channel assembly. The accessibility of sites to chaperones will depend on the degree of CFTR folding or unfolding. Different folded states may be recognized by unique combinations of HSC70, DNAJA1 and DNAJA2, leading to divergent biological effects.
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Affiliation(s)
- Imad Baaklini
- McGill University, Department of Biochemistry, Montreal, H3G 1Y6, Canada
| | | | - Gergely L Lukacs
- McGill University, Department of Biochemistry, Montreal, H3G 1Y6, Canada.,McGill University, Department of Physiology, Montreal, H3G 1Y6, Canada
| | - Jason C Young
- McGill University, Department of Biochemistry, Montreal, H3G 1Y6, Canada.
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Kleizen B, Hunt JF, Callebaut I, Hwang TC, Sermet-Gaudelus I, Hafkemeyer S, Sheppard DN. CFTR: New insights into structure and function and implications for modulation by small molecules. J Cyst Fibros 2020; 19 Suppl 1:S19-S24. [DOI: 10.1016/j.jcf.2019.10.021] [Citation(s) in RCA: 10] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/31/2019] [Revised: 10/17/2019] [Accepted: 10/18/2019] [Indexed: 12/22/2022]
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40
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Froux L, Elbahnsi A, Boucherle B, Billet A, Baatallah N, Hoffmann B, Alliot J, Zelli R, Zeinyeh W, Haudecoeur R, Chevalier B, Fortuné A, Mirval S, Simard C, Lehn P, Mornon JP, Hinzpeter A, Becq F, Callebaut I, Décout JL. Targeting different binding sites in the CFTR structures allows to synergistically potentiate channel activity. Eur J Med Chem 2020; 190:112116. [DOI: 10.1016/j.ejmech.2020.112116] [Citation(s) in RCA: 6] [Impact Index Per Article: 1.2] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/16/2019] [Revised: 01/24/2020] [Accepted: 02/03/2020] [Indexed: 02/06/2023]
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Farkas B, Tordai H, Padányi R, Tordai A, Gera J, Paragi G, Hegedűs T. Discovering the chloride pathway in the CFTR channel. Cell Mol Life Sci 2020; 77:765-778. [PMID: 31327045 PMCID: PMC7039865 DOI: 10.1007/s00018-019-03211-4] [Citation(s) in RCA: 11] [Impact Index Per Article: 2.2] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/22/2019] [Revised: 06/22/2019] [Accepted: 06/26/2019] [Indexed: 12/15/2022]
Abstract
Cystic fibrosis (CF), a lethal monogenic disease, is caused by pathogenic variants of the CFTR chloride channel. The majority of CF mutations affect protein folding and stability leading overall to diminished apical anion conductance of epithelial cells. The recently published cryo-EM structures of full-length human and zebrafish CFTR provide a good model to gain insight into structure-function relationships of CFTR variants. Although, some of the structures were determined in the phosphorylated and ATP-bound active state, none of the static structures showed an open pathway for chloride permeation. Therefore, we performed molecular dynamics simulations to generate a conformational ensemble of the protein and used channel detecting algorithms to identify conformations with an opened channel. Our simulations indicate a main intracellular entry at TM4/6, a secondary pore at TM10/12, and a bottleneck region involving numerous amino acids from TM1, TM6, and TM12 in accordance with experiments. Since chloride ions entered the pathway in our equilibrium simulations, but did not traverse the bottleneck region, we performed metadynamics simulations, which revealed two possible exits. One of the chloride ions exits includes hydrophobic lipid tails that may explain the lipid-dependency of CFTR function. In summary, our in silico study provides a detailed description of a potential chloride channel pathway based on a recent cryo-EM structure and may help to understand the gating of the CFTR chloride channel, thus contributing to novel strategies to rescue dysfunctional mutants.
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Affiliation(s)
- Bianka Farkas
- Department of Biophysics and Radiation Biology, Semmelweis University, Budapest, Hungary
- Faculty of Information Technology, Pázmány Péter Catholic University, Budapest, Hungary
| | - Hedvig Tordai
- Department of Biophysics and Radiation Biology, Semmelweis University, Budapest, Hungary
| | - Rita Padányi
- Department of Biophysics and Radiation Biology, Semmelweis University, Budapest, Hungary
- MTA-SE Molecular Biophysics Research Group, Hungarian Academy of Sciences, Budapest, Hungary
| | - Attila Tordai
- Department of Pathophysiology, Semmelweis University, Budapest, Hungary
| | - János Gera
- Department of Medical Chemistry, University of Szeged, Szeged, Hungary
| | - Gábor Paragi
- MTA-SZTE Biomimetic System Research Group, Hungarian Academy of Sciences, Szeged, Hungary
- Institute of Physics, University of Pécs, Pecs, Hungary
| | - Tamás Hegedűs
- Department of Biophysics and Radiation Biology, Semmelweis University, Budapest, Hungary.
- MTA-SE Molecular Biophysics Research Group, Hungarian Academy of Sciences, Budapest, Hungary.
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42
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Cui G, Hong J, Chung-Davidson YW, Infield D, Xu X, Li J, Simhaev L, Khazanov N, Stauffer B, Imhoff B, Cottrill K, Blalock JE, Li W, Senderowitz H, Sorscher E, McCarty NA, Gaggar A. An Ancient CFTR Ortholog Informs Molecular Evolution in ABC Transporters. Dev Cell 2019; 51:421-430.e3. [PMID: 31679858 DOI: 10.1016/j.devcel.2019.09.017] [Citation(s) in RCA: 16] [Impact Index Per Article: 2.7] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/18/2018] [Revised: 07/30/2019] [Accepted: 09/24/2019] [Indexed: 01/13/2023]
Abstract
The cystic fibrosis transmembrane conductance regulator (CFTR) is a chloride channel central to the development of secretory diarrhea and cystic fibrosis. The oldest CFTR ortholog identified is from dogfish shark, which retains similar structural and functional characteristics to the mammalian protein, thereby highlighting CFTR's critical role in regulating epithelial ion transport in vertebrates. However, the identification of an early CFTR ortholog with altered structure or function would provide critical insight into the evolution of epithelial anion transport. Here, we describe the earliest known CFTR, expressed in sea lamprey (Petromyzon marinus), with unique structural features, altered kinetics of activation and sensitivity to inhibition, and altered single-channel conductance compared to human CFTR. Our data provide the earliest evolutionary evidence of CFTR, offering insight regarding changes in gene and protein structure that underpin evolution from transporter to anion channel. Importantly, these data provide a unique platform to enhance our understanding of vertebrate phylogeny over a critical period of evolutionary expansion.
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Affiliation(s)
- Guiying Cui
- Department of Pediatrics and Children's Healthcare of Atlanta, Center for Cystic Fibrosis and Airways Disease Research, Emory University, Atlanta, GA 30322, USA
| | - Jeong Hong
- Department of Pediatrics and Children's Healthcare of Atlanta, Center for Cystic Fibrosis and Airways Disease Research, Emory University, Atlanta, GA 30322, USA; Department of Medicine, Gregory Fleming James Cystic Fibrosis Research Center, and Program in Protease and Matrix Biology, University of Alabama at Birmingham, Birmingham, AL 35294, USA
| | - Yu-Wen Chung-Davidson
- Department of Fisheries and Wildlife, Michigan State University, East Lansing, MI 48823, USA
| | - Daniel Infield
- Department of Pediatrics and Children's Healthcare of Atlanta, Center for Cystic Fibrosis and Airways Disease Research, Emory University, Atlanta, GA 30322, USA; Department of Molecular Physiology and Biophysics, University of Iowa, Iowa City, IA 52242, USA
| | - Xin Xu
- Department of Medicine, Gregory Fleming James Cystic Fibrosis Research Center, and Program in Protease and Matrix Biology, University of Alabama at Birmingham, Birmingham, AL 35294, USA; Birmingham Veterans Administration Medical Center, Birmingham, AL 35233, USA
| | - Jindong Li
- Department of Medicine, Gregory Fleming James Cystic Fibrosis Research Center, and Program in Protease and Matrix Biology, University of Alabama at Birmingham, Birmingham, AL 35294, USA; Birmingham Veterans Administration Medical Center, Birmingham, AL 35233, USA
| | - Luba Simhaev
- Department of Chemistry, Bar-Ilan University, Ramat Gan, Israel
| | - Netaly Khazanov
- Department of Chemistry, Bar-Ilan University, Ramat Gan, Israel
| | - Brandon Stauffer
- Department of Pediatrics and Children's Healthcare of Atlanta, Center for Cystic Fibrosis and Airways Disease Research, Emory University, Atlanta, GA 30322, USA
| | - Barry Imhoff
- Department of Pediatrics and Children's Healthcare of Atlanta, Center for Cystic Fibrosis and Airways Disease Research, Emory University, Atlanta, GA 30322, USA
| | - Kirsten Cottrill
- Department of Pediatrics and Children's Healthcare of Atlanta, Center for Cystic Fibrosis and Airways Disease Research, Emory University, Atlanta, GA 30322, USA
| | - J Edwin Blalock
- Department of Medicine, Gregory Fleming James Cystic Fibrosis Research Center, and Program in Protease and Matrix Biology, University of Alabama at Birmingham, Birmingham, AL 35294, USA
| | - Weiming Li
- Department of Fisheries and Wildlife, Michigan State University, East Lansing, MI 48823, USA
| | | | - Eric Sorscher
- Department of Pediatrics and Children's Healthcare of Atlanta, Center for Cystic Fibrosis and Airways Disease Research, Emory University, Atlanta, GA 30322, USA; Department of Medicine, Gregory Fleming James Cystic Fibrosis Research Center, and Program in Protease and Matrix Biology, University of Alabama at Birmingham, Birmingham, AL 35294, USA
| | - Nael A McCarty
- Department of Pediatrics and Children's Healthcare of Atlanta, Center for Cystic Fibrosis and Airways Disease Research, Emory University, Atlanta, GA 30322, USA
| | - Amit Gaggar
- Department of Medicine, Gregory Fleming James Cystic Fibrosis Research Center, and Program in Protease and Matrix Biology, University of Alabama at Birmingham, Birmingham, AL 35294, USA; Birmingham Veterans Administration Medical Center, Birmingham, AL 35233, USA.
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Abreu B, Lopes EF, Oliveira ASF, Soares CM. F508del disturbs the dynamics of the nucleotide binding domains of CFTR before and after ATP hydrolysis. Proteins 2019; 88:113-126. [PMID: 31298435 DOI: 10.1002/prot.25776] [Citation(s) in RCA: 12] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Key Words] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/11/2019] [Revised: 06/17/2019] [Accepted: 07/06/2019] [Indexed: 12/20/2022]
Abstract
The cystic fibrosis transmembrane conductance regulator (CFTR) channel is an ion channel responsible for chloride transport in epithelia and it belongs to the class of ABC transporters. The deletion of phenylalanine 508 (F508del) in CFTR is the most common mutation responsible for cystic fibrosis. Little is known about the effect of the mutation in the isolated nucleotide binding domains (NBDs), on dimer dynamics, ATP hydrolysis and even on nucleotide binding. Using molecular dynamics simulations of the human CFTR NBD dimer, we showed that F508del increases, in the prehydrolysis state, the inter-motif distance in both ATP binding sites (ABP) when ATP is bound. Additionally, a decrease in the number of catalytically competent conformations was observed in the presence of F508del. We used the subtraction technique to study the first 300 ps after ATP hydrolysis in the catalytic competent site and found that the F508del dimer evidences lower conformational changes than the wild type. Using longer simulation times, the magnitude of the conformational changes in both forms increases. Nonetheless, the F508del dimer shows lower C-α RMS values in comparison to the wild-type, on the F508del loop, on the residues surrounding the catalytic site and the portion of NBD2 adjacent to ABP1. These results provide evidence that F508del interferes with the NBD dynamics before and after ATP hydrolysis. These findings shed a new light on the effect of F508del on NBD dynamics and reveal a novel mechanism for the influence of F508del on CFTR.
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Affiliation(s)
- Bárbara Abreu
- Protein Modelling Lab, ITQB-NOVA, Universidade Nova de Lisboa, Oeiras, Portugal
| | - Emanuel F Lopes
- Protein Modelling Lab, ITQB-NOVA, Universidade Nova de Lisboa, Oeiras, Portugal
| | - A S F Oliveira
- Protein Modelling Lab, ITQB-NOVA, Universidade Nova de Lisboa, Oeiras, Portugal.,School of Biochemistry & Center for Computational Chemistry, University of Bristol, Bristol, UK
| | - Cláudio M Soares
- Protein Modelling Lab, ITQB-NOVA, Universidade Nova de Lisboa, Oeiras, Portugal
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44
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Beumer W, Swildens J, Leal T, Noel S, Anthonijsz H, van der Horst G, Kuiperij-Boersma H, Potman M, van Putten C, Biasutto P, Platenburg G, de Jonge H, Henig N, Ritsema T. Evaluation of eluforsen, a novel RNA oligonucleotide for restoration of CFTR function in in vitro and murine models of p.Phe508del cystic fibrosis. PLoS One 2019; 14:e0219182. [PMID: 31251792 PMCID: PMC6599119 DOI: 10.1371/journal.pone.0219182] [Citation(s) in RCA: 20] [Impact Index Per Article: 3.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/09/2019] [Accepted: 06/18/2019] [Indexed: 01/14/2023] Open
Abstract
Cystic fibrosis (CF) is caused by mutations in the gene encoding the epithelial chloride channel CF transmembrane conductance regulator (CFTR) protein. The most common mutation is a deletion of three nucleotides leading to the loss of phenylalanine at position 508 (p.Phe508del) in the protein. This study evaluates eluforsen, a novel, single-stranded, 33-nucleotide antisense oligonucleotide designed to restore CFTR function, in in vitro and in vivo models of p.Phe508del CF. The aims of the study were to demonstrate cellular uptake of eluforsen, and its efficacy in functional restoration of p.Phe508del-CFTR both in vitro and in vivo. In vitro, the effect of eluforsen was investigated in human CF pancreatic adenocarcinoma cells and human bronchial epithelial cells. Two mouse models were used to evaluate eluforsen in vivo. In vitro, eluforsen improved chloride efflux in CF pancreatic adenocarcinoma cell cultures and increased short-circuit current in primary human bronchial epithelial cells, both indicating restoration of CFTR function. In vivo, eluforsen was taken up by airway epithelium following oro-tracheal administration in mice, resulting in systemic exposure of eluforsen. In female F508del-CFTR mice, eluforsen significantly increased CFTR-mediated saliva secretion (used as a measure of CFTR function, equivalent to the sweat test in humans). Similarly, intranasal administration of eluforsen significantly improved nasal potential difference (NPD), and therefore CFTR conductance, in two CF mouse models. These findings indicate that eluforsen improved CFTR function in cell and animal models of p.Phe508del-CFTR-mediated CF and supported further development of eluforsen in human clinical trials, where eluforsen has also been shown to improve CFTR activity as measured by NPD.
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Affiliation(s)
- Wouter Beumer
- ProQR Therapeutics, Leiden, The Netherlands
- * E-mail:
| | | | - Teresinha Leal
- Université Catholique de Louvain, Louvain Centre for Toxicology and Applied Pharmacology, Brussels, Belgium
| | - Sabrina Noel
- Université Catholique de Louvain, Louvain Centre for Toxicology and Applied Pharmacology, Brussels, Belgium
| | | | | | | | | | | | | | | | - Hugo de Jonge
- Department of Gastroenterology and Hepatology, Erasmus University Medical Center, Rotterdam, The Netherlands
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45
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Sigoillot M, Overtus M, Grodecka M, Scholl D, Garcia-Pino A, Laeremans T, He L, Pardon E, Hildebrandt E, Urbatsch I, Steyaert J, Riordan JR, Govaerts C. Domain-interface dynamics of CFTR revealed by stabilizing nanobodies. Nat Commun 2019; 10:2636. [PMID: 31201318 PMCID: PMC6572788 DOI: 10.1038/s41467-019-10714-y] [Citation(s) in RCA: 20] [Impact Index Per Article: 3.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/13/2018] [Accepted: 05/21/2019] [Indexed: 01/17/2023] Open
Abstract
The leading cause of cystic fibrosis (CF) is the deletion of phenylalanine 508 (F508del) in the first nucleotide-binding domain (NBD1) of the cystic fibrosis transmembrane conductance regulator (CFTR). The mutation affects the thermodynamic stability of the domain and the integrity of the interface between NBD1 and the transmembrane domain leading to its clearance by the quality control system. Here, we develop nanobodies targeting NBD1 of human CFTR and demonstrate their ability to stabilize both isolated NBD1 and full-length protein. Crystal structures of NBD1-nanobody complexes provide an atomic description of the epitopes and reveal the molecular basis for stabilization. Furthermore, our data uncover a conformation of CFTR, involving detachment of NBD1 from the transmembrane domain, which contrast with the compact assembly observed in cryo-EM structures. This unexpected interface rearrangement is likely to have major relevance for CF pathogenesis but also for the normal function of CFTR and other ABC proteins.
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Affiliation(s)
- Maud Sigoillot
- SFMB, Université Libre de Bruxelles (ULB), CP206/02, Boulevard du Triomphe, building BC, B-1050, Brussels, Belgium
| | - Marie Overtus
- SFMB, Université Libre de Bruxelles (ULB), CP206/02, Boulevard du Triomphe, building BC, B-1050, Brussels, Belgium
| | - Magdalena Grodecka
- SFMB, Université Libre de Bruxelles (ULB), CP206/02, Boulevard du Triomphe, building BC, B-1050, Brussels, Belgium
| | - Daniel Scholl
- SFMB, Université Libre de Bruxelles (ULB), CP206/02, Boulevard du Triomphe, building BC, B-1050, Brussels, Belgium
| | - Abel Garcia-Pino
- Laboratoire de Microbiologie Moléculaire et Cellulaire, ULB CP300, rue des Professeurs Jeener et Brachet 12, B-6041, Charleroi, Belgium
| | - Toon Laeremans
- Structural Biology Brussels, Vrije Universiteit Brussel (VUB), Pleinlaan 2, B-1050, Brussels, Belgium.,VIB-VUB center for Structural Biology, VIB, Pleinlaan 2, B-1050, Brussels, Belgium
| | - Lihua He
- Department of Biochemistry and Biophysics and Cystic Fibrosis Center, University of North Carolina-Chapel Hill, Chapel Hill, NC, 27599, USA
| | - Els Pardon
- Structural Biology Brussels, Vrije Universiteit Brussel (VUB), Pleinlaan 2, B-1050, Brussels, Belgium.,VIB-VUB center for Structural Biology, VIB, Pleinlaan 2, B-1050, Brussels, Belgium
| | - Ellen Hildebrandt
- Department of Cell Biology and Biochemistry and Center for Membrane Protein Research, Texas Tech University Health Sciences Center, 3601 4th Street, Stop 6540, Lubbock, TX, 79430, USA
| | - Ina Urbatsch
- Department of Cell Biology and Biochemistry and Center for Membrane Protein Research, Texas Tech University Health Sciences Center, 3601 4th Street, Stop 6540, Lubbock, TX, 79430, USA
| | - Jan Steyaert
- Structural Biology Brussels, Vrije Universiteit Brussel (VUB), Pleinlaan 2, B-1050, Brussels, Belgium.,VIB-VUB center for Structural Biology, VIB, Pleinlaan 2, B-1050, Brussels, Belgium
| | - John R Riordan
- Department of Biochemistry and Biophysics and Cystic Fibrosis Center, University of North Carolina-Chapel Hill, Chapel Hill, NC, 27599, USA
| | - Cedric Govaerts
- SFMB, Université Libre de Bruxelles (ULB), CP206/02, Boulevard du Triomphe, building BC, B-1050, Brussels, Belgium.
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46
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Marinko J, Huang H, Penn WD, Capra JA, Schlebach JP, Sanders CR. Folding and Misfolding of Human Membrane Proteins in Health and Disease: From Single Molecules to Cellular Proteostasis. Chem Rev 2019; 119:5537-5606. [PMID: 30608666 PMCID: PMC6506414 DOI: 10.1021/acs.chemrev.8b00532] [Citation(s) in RCA: 180] [Impact Index Per Article: 30.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/25/2018] [Indexed: 12/13/2022]
Abstract
Advances over the past 25 years have revealed much about how the structural properties of membranes and associated proteins are linked to the thermodynamics and kinetics of membrane protein (MP) folding. At the same time biochemical progress has outlined how cellular proteostasis networks mediate MP folding and manage misfolding in the cell. When combined with results from genomic sequencing, these studies have established paradigms for how MP folding and misfolding are linked to the molecular etiologies of a variety of diseases. This emerging framework has paved the way for the development of a new class of small molecule "pharmacological chaperones" that bind to and stabilize misfolded MP variants, some of which are now in clinical use. In this review, we comprehensively outline current perspectives on the folding and misfolding of integral MPs as well as the mechanisms of cellular MP quality control. Based on these perspectives, we highlight new opportunities for innovations that bridge our molecular understanding of the energetics of MP folding with the nuanced complexity of biological systems. Given the many linkages between MP misfolding and human disease, we also examine some of the exciting opportunities to leverage these advances to address emerging challenges in the development of therapeutics and precision medicine.
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Affiliation(s)
- Justin
T. Marinko
- Department
of Biochemistry, Vanderbilt University, Nashville, Tennessee 37240, United States
- Center
for Structural Biology, Vanderbilt University, Nashville, Tennessee 37240, United States
| | - Hui Huang
- Department
of Biochemistry, Vanderbilt University, Nashville, Tennessee 37240, United States
- Center
for Structural Biology, Vanderbilt University, Nashville, Tennessee 37240, United States
| | - Wesley D. Penn
- Department
of Chemistry, Indiana University, Bloomington, Indiana 47405, United States
| | - John A. Capra
- Center
for Structural Biology, Vanderbilt University, Nashville, Tennessee 37240, United States
- Department
of Biological Sciences, Vanderbilt University, Nashville, Tennessee 37245, United States
| | - Jonathan P. Schlebach
- Department
of Chemistry, Indiana University, Bloomington, Indiana 47405, United States
| | - Charles R. Sanders
- Department
of Biochemistry, Vanderbilt University, Nashville, Tennessee 37240, United States
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47
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Strickland KM, Stock G, Cui G, Hwang H, Infield DT, Schmidt-Krey I, McCarty NA, Gumbart JC. ATP-Dependent Signaling in Simulations of a Revised Model of Cystic Fibrosis Transmembrane Conductance Regulator (CFTR). J Phys Chem B 2019; 123:3177-3188. [PMID: 30921517 DOI: 10.1021/acs.jpcb.8b11970] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/17/2022]
Abstract
Cystic fibrosis transmembrane conductance regulator (CFTR) is a member of the ATP-binding cassette (ABC) transporter superfamily that has uniquely evolved to function as a chloride channel. It binds and hydrolyzes ATP at its nucleotide binding domains to form a pore providing a diffusive pathway within its transmembrane domains. CFTR is the only known protein from the ABC superfamily with channel activity, and its dysfunction causes the disease cystic fibrosis. While much is known about the functional aspects of CFTR, significant gaps remain, such as the structure-function relationship underlying signaling of ATP binding. In the present work, we refined an existing homology model using an intermediate-resolution (9 Å) published cryo-electron microscopy map. The newly derived models have been simulated in equilibrium molecular dynamics simulations for a total of 2.5 μs in multiple ATP-occupancy states. Putative conformational movements connecting ATP binding with pore formation are elucidated and quantified. Additionally, new interdomain interactions between E543, K968, and K1292 have been identified and confirmed experimentally; these interactions may be relevant for signaling ATP binding and hydrolysis to the transmembrane domains and induction of pore opening.
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Affiliation(s)
- Kerry M Strickland
- School of Chemistry and Biochemistry , Georgia Institute of Technology , Atlanta , Georgia 30332 , United States
| | - Gorman Stock
- School of Chemistry and Biochemistry , Georgia Institute of Technology , Atlanta , Georgia 30332 , United States
| | - Guiying Cui
- Division of Pulmonology, Allergy and Immunology, Cystic Fibrosis, and Sleep, Department of Pediatrics, Center for Cystic Fibrosis and Airways Disease Research, Emory+Children's Pediatric Research Center , Emory University School of Medicine and Children's Healthcare of Atlanta , Atlanta , Georgia 30322 , United States
| | - Hyea Hwang
- School of Materials Science and Engineering , Georgia Institute of Technology , Atlanta , Georgia 30332 , United States
| | - Daniel T Infield
- Division of Pulmonology, Allergy and Immunology, Cystic Fibrosis, and Sleep, Department of Pediatrics, Center for Cystic Fibrosis and Airways Disease Research, Emory+Children's Pediatric Research Center , Emory University School of Medicine and Children's Healthcare of Atlanta , Atlanta , Georgia 30322 , United States
| | - Ingeborg Schmidt-Krey
- School of Chemistry and Biochemistry , Georgia Institute of Technology , Atlanta , Georgia 30332 , United States.,School of Biological Sciences , Georgia Institute of Technology , Atlanta , Georgia 30332 , United States.,Parker H. Petit Institute for Bioengineering and Bioscience , Georgia Institute of Technology , Atlanta , Georgia 30332 , United States
| | - Nael A McCarty
- Division of Pulmonology, Allergy and Immunology, Cystic Fibrosis, and Sleep, Department of Pediatrics, Center for Cystic Fibrosis and Airways Disease Research, Emory+Children's Pediatric Research Center , Emory University School of Medicine and Children's Healthcare of Atlanta , Atlanta , Georgia 30322 , United States.,Parker H. Petit Institute for Bioengineering and Bioscience , Georgia Institute of Technology , Atlanta , Georgia 30332 , United States
| | - James C Gumbart
- School of Chemistry and Biochemistry , Georgia Institute of Technology , Atlanta , Georgia 30332 , United States.,School of Biological Sciences , Georgia Institute of Technology , Atlanta , Georgia 30332 , United States.,Parker H. Petit Institute for Bioengineering and Bioscience , Georgia Institute of Technology , Atlanta , Georgia 30332 , United States.,School of Physics , Georgia Institute of Technology , Atlanta , Georgia 30332 , United States
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48
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Doiron JE, Le CA, Ody BK, Brace JB, Post SJ, Thacker NL, Hill HM, Breton GW, Mulder MJ, Chang S, Bridges TM, Tang L, Wang W, Rowe SM, Aller SG, Turlington M. Evaluation of 1,2,3-Triazoles as Amide Bioisosteres In Cystic Fibrosis Transmembrane Conductance Regulator Modulators VX-770 and VX-809. Chemistry 2019; 25:3662-3674. [PMID: 30650214 PMCID: PMC6469399 DOI: 10.1002/chem.201805919] [Citation(s) in RCA: 16] [Impact Index Per Article: 2.7] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/27/2018] [Revised: 01/14/2019] [Indexed: 12/25/2022]
Abstract
The 1,2,3-triazole has been successfully utilized as an amide bioisostere in multiple therapeutic contexts. Based on this precedent, triazole analogues derived from VX-809 and VX-770, prominent amide-containing modulators of the cystic fibrosis transmembrane conductance regulator (CFTR), were synthesized and evaluated for CFTR modulation. Triazole 11, derived from VX-809, displayed markedly reduced efficacy in F508del-CFTR correction in cellular TECC assays in comparison to VX-809. Surprisingly, triazole analogues derived from potentiator VX-770 displayed no potentiation of F508del, G551D, or WT-CFTR in cellular Ussing chamber assays. However, patch clamp analysis revealed that triazole 60 potentiates WT-CFTR similarly to VX-770. The efficacy of 60 in the cell-free patch clamp experiment suggests that the loss of activity in the cellular assay could be due to the inability of VX-770 triazole derivatives to reach the CFTR binding site. Moreover, in addition to the negative impact on biological activity, triazoles in both structural classes displayed decreased metabolic stability in human microsomes relative to the analogous amides. In contrast to the many studies that demonstrate the advantages of using the 1,2,3-triazole, these findings highlight the negative impacts that can arise from replacement of the amide with the triazole and suggest that caution is warranted when considering use of the 1,2,3-triazole as an amide bioisostere.
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Affiliation(s)
- Jake E. Doiron
- Department of Chemistry and Biochemistry, Berry College, Mount Berry, Georgia 30165 (USA),
| | - Christina A. Le
- Department of Pharmacology and Toxicology, University of Alabama at Birmingham, Birmingham, Alabama 35205 (USA),
| | - Britton K. Ody
- Department of Chemistry and Biochemistry, Berry College, Mount Berry, Georgia 30165 (USA),
| | - Jonathon B. Brace
- Department of Chemistry and Biochemistry, Berry College, Mount Berry, Georgia 30165 (USA),
| | - Savannah J. Post
- Department of Chemistry and Biochemistry, Berry College, Mount Berry, Georgia 30165 (USA),
| | - Nathan L. Thacker
- Department of Chemistry and Biochemistry, Berry College, Mount Berry, Georgia 30165 (USA),
| | - Harrison M. Hill
- Department of Chemistry and Biochemistry, Berry College, Mount Berry, Georgia 30165 (USA),
| | - Gary W. Breton
- Department of Chemistry and Biochemistry, Berry College, Mount Berry, Georgia 30165 (USA),
| | - Matthew J. Mulder
- Vanderbilt Center for Neuroscience Drug Discovery, Nashville, Tennessee 37232 (USA)
| | - Sichen Chang
- Vanderbilt Center for Neuroscience Drug Discovery, Nashville, Tennessee 37232 (USA)
| | - Thomas M. Bridges
- Vanderbilt Center for Neuroscience Drug Discovery, Nashville, Tennessee 37232 (USA)
| | - Liping Tang
- Departments of Medicine and Pediatrics, University of Alabama at Birmingham, Birmingham, Alabama 35205 (USA)
- Gregory Fleming James Cystic Fibrosis Research Center, University of Alabama at Birmingham, Birmingham, Alabama 35205 (USA)
| | - Wei Wang
- Gregory Fleming James Cystic Fibrosis Research Center, University of Alabama at Birmingham, Birmingham, Alabama 35205 (USA)
- Department of Cell, Development, and Integrative Biology, University of Alabama at Birmingham, Birmingham, Alabama 35205 (USA)
| | - Steven M. Rowe
- Departments of Medicine and Pediatrics, University of Alabama at Birmingham, Birmingham, Alabama 35205 (USA)
- Gregory Fleming James Cystic Fibrosis Research Center, University of Alabama at Birmingham, Birmingham, Alabama 35205 (USA)
- Department of Cell, Development, and Integrative Biology, University of Alabama at Birmingham, Birmingham, Alabama 35205 (USA)
| | - Stephen G. Aller
- Department of Pharmacology and Toxicology, University of Alabama at Birmingham, Birmingham, Alabama 35205 (USA),
| | - Mark Turlington
- Department of Chemistry and Biochemistry, Berry College, Mount Berry, Georgia 30165 (USA),
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49
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Csanády L, Vergani P, Gadsby DC. STRUCTURE, GATING, AND REGULATION OF THE CFTR ANION CHANNEL. Physiol Rev 2019; 99:707-738. [PMID: 30516439 DOI: 10.1152/physrev.00007.2018] [Citation(s) in RCA: 164] [Impact Index Per Article: 27.3] [Reference Citation Analysis] [Abstract] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/17/2022] Open
Abstract
The cystic fibrosis transmembrane conductance regulator (CFTR) belongs to the ATP binding cassette (ABC) transporter superfamily but functions as an anion channel crucial for salt and water transport across epithelial cells. CFTR dysfunction, because of mutations, causes cystic fibrosis (CF). The anion-selective pore of the CFTR protein is formed by its two transmembrane domains (TMDs) and regulated by its cytosolic domains: two nucleotide binding domains (NBDs) and a regulatory (R) domain. Channel activation requires phosphorylation of the R domain by cAMP-dependent protein kinase (PKA), and pore opening and closing (gating) of phosphorylated channels is driven by ATP binding and hydrolysis at the NBDs. This review summarizes available information on structure and mechanism of the CFTR protein, with a particular focus on atomic-level insight gained from recent cryo-electron microscopic structures and on the molecular mechanisms of channel gating and its regulation. The pharmacological mechanisms of small molecules targeting CFTR's ion channel function, aimed at treating patients suffering from CF and other diseases, are briefly discussed.
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Affiliation(s)
- László Csanády
- Department of Medical Biochemistry, Semmelweis University , Budapest , Hungary ; MTA-SE Ion Channel Research Group, Budapest , Hungary ; Department of Neuroscience, Physiology and Pharmacology, University College London , London , United Kingdom ; and Laboratory of Cardiac/Membrane Physiology, The Rockefeller University , New York, New York
| | - Paola Vergani
- Department of Medical Biochemistry, Semmelweis University , Budapest , Hungary ; MTA-SE Ion Channel Research Group, Budapest , Hungary ; Department of Neuroscience, Physiology and Pharmacology, University College London , London , United Kingdom ; and Laboratory of Cardiac/Membrane Physiology, The Rockefeller University , New York, New York
| | - David C Gadsby
- Department of Medical Biochemistry, Semmelweis University , Budapest , Hungary ; MTA-SE Ion Channel Research Group, Budapest , Hungary ; Department of Neuroscience, Physiology and Pharmacology, University College London , London , United Kingdom ; and Laboratory of Cardiac/Membrane Physiology, The Rockefeller University , New York, New York
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50
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Chen X, Zhu S, Zhenin M, Xu W, Bose SJ, Wong MPF, Leung GPH, Senderowitz H, Chen JH. A defective flexible loop contributes to the processing and gating defects of the predominant cystic fibrosis-causing mutation. FASEB J 2019; 33:5126-5142. [PMID: 30668920 DOI: 10.1096/fj.201801218rr] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/11/2022]
Abstract
People with the genetic disease cystic fibrosis (CF) often carry a deletion mutation ΔF508 on the gene encoding the CF transmembrane conductance regulator (CFTR) Cl- channel. This mutation greatly reduces the CFTR maturation process and slows the channel opening rate. Here, we investigate whether residues near F508 contribute to these defects in ΔF508-CFTR. Most deletion mutations, but not alanine substitutions, of individual residues from positions 503 to 513 impaired CFTR maturation. Interestingly, only protein processing of ΔY512-CFTR, like that of ΔF508-CFTR, was greatly improved by low-temperature culture at 27°C or small-molecule corrector C18. The 2 mutant Cl- channels were equally slow to open, suggesting that they may share common structural flaws. Studies on the H3-H4 loop that links residues F508 and Y512 demonstrate that G509A/V510G mutations, moving G509 1 position backward in the loop, markedly enhanced ΔF508-CFTR maturation and opening rate while promoting protein stability and persistence of the H3 helix in ΔF508 nucleotide-binding domain 1. Moreover, V510A/S511A mutations noticeably increased ΔY512-CFTR maturation at 27°C and its opening rate. Thus, loop abnormalities may contribute to ΔF508- and ΔY512-CFTR defects. Importantly, correcting defects from G509 displacement in ΔF508-CFTR may offer a new avenue for drug discovery and CF treatments.-Chen, X., Zhu, S., Zhenin, M., Xu, W., Bose, S. J., Wong, M. P.-F., Leung, G. P. H., Senderowitz, H., Chen, J.-H. A defective flexible loop contributes to the processing and gating defects of the predominant cystic fibrosis-causing mutation.
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Affiliation(s)
- Xinying Chen
- School of Biomedical Sciences, University of Hong Kong, Hong Kong.,The University of Hong Kong Shenzhen Institute of Research and Innovation, Shenzhen, China
| | - Siyu Zhu
- School of Biomedical Sciences, University of Hong Kong, Hong Kong.,The University of Hong Kong Shenzhen Institute of Research and Innovation, Shenzhen, China
| | - Michael Zhenin
- Department of Chemistry, Bar Ilan University, Ramat-Gan, Israel
| | - Weiyi Xu
- School of Biomedical Sciences, University of Hong Kong, Hong Kong.,The University of Hong Kong Shenzhen Institute of Research and Innovation, Shenzhen, China
| | - Samuel J Bose
- School of Physiology, Pharmacology, and Neuroscience, University of Bristol, Bristol, United Kingdom; and
| | - Molly Pik-Fan Wong
- School of Biomedical Sciences, University of Hong Kong, Hong Kong.,The University of Hong Kong Shenzhen Institute of Research and Innovation, Shenzhen, China
| | - George P H Leung
- Department of Pharmacology and Pharmacy, University of Hong Kong, Hong Kong, China
| | | | - Jeng-Haur Chen
- School of Biomedical Sciences, University of Hong Kong, Hong Kong.,The University of Hong Kong Shenzhen Institute of Research and Innovation, Shenzhen, China
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