Research

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22 pages, 5210 KiB

Open AccessArticle

Transmembrane Helices 7 and 8 Confer Aggregation Sensitivity to the Cystic Fibrosis Transmembrane Conductance Regulator

by Bertrand Kleizen, Eduardo de Mattos, Olga Papaioannou, Michele Monti, Gian Gaetano Tartaglia, Peter van der Sluijs and Ineke Braakman

Int. J. Mol. Sci. 2023, 24(21), 15741; https://doi.org/10.3390/ijms242115741 - 30 Oct 2023

Viewed by 901

Abstract

The Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) is a large multi-spanning membrane protein that is susceptible to misfolding and aggregation. We have identified here the region responsible for this instability. Temperature-induced aggregation of C-terminally truncated versions of CFTR demonstrated that all truncations up [...] Read more.

The Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) is a large multi-spanning membrane protein that is susceptible to misfolding and aggregation. We have identified here the region responsible for this instability. Temperature-induced aggregation of C-terminally truncated versions of CFTR demonstrated that all truncations up to the second transmembrane domain (TMD2), including the R region, largely resisted aggregation. Limited proteolysis identified a folded structure that was prone to aggregation and consisted of TMD2 and at least part of the Regulatory Region R. Only when both TM7 (TransMembrane helix 7) and TM8 were present, TMD2 fragments became as aggregation-sensitive as wild-type CFTR, in line with increased thermo-instability of late CFTR nascent chains and in silico prediction of aggregation propensity. In accord, isolated TMD2 was degraded faster in cells than isolated TMD1. We conclude that TMD2 extended at its N-terminus with part of the R region forms a protease-resistant structure that induces heat instability in CFTR and may be responsible for its limited intracellular stability. Full article

(This article belongs to the Special Issue Structure-Based Understanding of the Function-Dysfunction of ABC Transporters)

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15 pages, 3800 KiB

Open AccessArticle

Structural and Pathogenic Impacts of ABCA4 Variants in Retinal Degenerations—An In-Silico Study

by Senem Cevik, Subhasis B. Biswas and Esther E. Biswas-Fiss

Int. J. Mol. Sci. 2023, 24(8), 7280; https://doi.org/10.3390/ijms24087280 - 14 Apr 2023

Cited by 2 | Viewed by 1832

Abstract

The retina-specific ATP-binding cassette transporter protein ABCA4 is responsible for properly continuing the visual cycle by removing toxic retinoid byproducts of phototransduction. Functional impairment caused by ABCA4 sequence variations is the leading cause of autosomal recessive inherited retinal disorders, including Stargardt disease, retinitis [...] Read more.

The retina-specific ATP-binding cassette transporter protein ABCA4 is responsible for properly continuing the visual cycle by removing toxic retinoid byproducts of phototransduction. Functional impairment caused by ABCA4 sequence variations is the leading cause of autosomal recessive inherited retinal disorders, including Stargardt disease, retinitis pigmentosa, and cone-rod dystrophy. To date, more than 3000 ABCA4 genetic variants have been identified, approximately 40 percent of which have not been able to be classified for pathogenicity assessments. This study examined 30 missense ABCA4 variants using AlphaFold2 protein modeling and computational structure analysis for pathogenicity prediction. All variants classified as pathogenic (n = 10) were found to have deleterious structural consequences. Eight of the ten benign variants were structurally neutral, while the remaining two resulted in mild structural changes. This study’s results provided multiple lines of computational pathogenicity evidence for eight ABCA4 variants of uncertain clinical significance. Overall, in silico analyses of ABCA4 can provide a valuable tool for understanding the molecular mechanisms of retinal degeneration and their pathogenic impact. Full article

(This article belongs to the Special Issue Structure-Based Understanding of the Function-Dysfunction of ABC Transporters)

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18 pages, 5969 KiB

Open AccessArticle

Identification and Empiric Evaluation of New Inhibitors of the Multidrug Transporter P-Glycoprotein (ABCB1)

by Yasmeen Cheema, Yusra Sajid Kiani, Kenneth J. Linton and Ishrat Jabeen

Int. J. Mol. Sci. 2023, 24(6), 5298; https://doi.org/10.3390/ijms24065298 - 10 Mar 2023

Cited by 4 | Viewed by 1541

Abstract

The expression of the drug efflux pump ABCB1 correlates negatively with cancer survival, making the transporter an attractive target for therapeutic inhibition. In order to identify new inhibitors of ABCB1, we have exploited the cryo-EM structure of the protein to develop a pharmacophore [...] Read more.

The expression of the drug efflux pump ABCB1 correlates negatively with cancer survival, making the transporter an attractive target for therapeutic inhibition. In order to identify new inhibitors of ABCB1, we have exploited the cryo-EM structure of the protein to develop a pharmacophore model derived from the best docked conformations of a structurally diverse range of known inhibitors. The pharmacophore model was used to screen the Chembridge compound library. We identified six new potential inhibitors with distinct chemistry compared to the third-generation inhibitor tariquidar and with favourable lipophilic efficiency (LipE) and lipophilicity (CLogP) characteristics, suggesting oral bioavailability. These were evaluated experimentally for efficacy and potency using a fluorescent drug transport assay in live cells. The half-maximal inhibitory concentrations (IC₅₀) of four of the compounds were in the low nanomolar range (1.35 to 26.4 nM). The two most promising compounds were also able to resensitise ABCB1-expressing cells to taxol. This study demonstrates the utility of cryo-electron microscopy structure determination for drug identification and design. Full article

(This article belongs to the Special Issue Structure-Based Understanding of the Function-Dysfunction of ABC Transporters)

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13 pages, 2060 KiB

Open AccessArticle

Ivacaftor-Mediated Potentiation of ABCB4 Missense Mutations Affecting Critical Motifs of the NBDs: Repositioning Perspectives for Hepatobiliary Diseases

by Jean-Louis Delaunay, Ahmad Elbahnsi, Alix Bruneau, Claire Madry, Anne-Marie Durand-Schneider, Anne Stary, Chantal Housset, Jérémie Gautheron, Isabelle Callebaut and Tounsia Aït-Slimane

Int. J. Mol. Sci. 2023, 24(2), 1236; https://doi.org/10.3390/ijms24021236 - 08 Jan 2023

Cited by 1 | Viewed by 1521

Abstract

ABCB4 (ATP-binding cassette subfamily B member 4) is a hepatocanalicular floppase involved in biliary phosphatidylcholine (PC) secretion. Variations in the ABCB4 gene give rise to several biliary diseases, including progressive familial intrahepatic cholestasis type 3 (PFIC3), an autosomal recessive disease that can be [...] Read more.

ABCB4 (ATP-binding cassette subfamily B member 4) is a hepatocanalicular floppase involved in biliary phosphatidylcholine (PC) secretion. Variations in the ABCB4 gene give rise to several biliary diseases, including progressive familial intrahepatic cholestasis type 3 (PFIC3), an autosomal recessive disease that can be lethal in the absence of liver transplantation. In this study, we investigated the effect and potential rescue of ten ABCB4 missense variations in NBD1:NBD2 homologous positions (Y403H/Y1043H, K435M/K1075M, E558K/E1200A, D564G/D1206G and H589Y/H1231Y) all localized at the conserved and functionally critical motifs of ABC transporters, six of which are mutated in patients. By combining structure analysis and in vitro studies, we found that all ten mutants were normally processed and localized at the canalicular membrane of HepG2 cells, but showed dramatically impaired PC transport activity that was significantly rescued by treatment with the clinically approved CFTR potentiator ivacaftor. Our results provide evidence that functional ABCB4 mutations are rescued by ivacaftor, paving the way for the repositioning of this potentiator for the treatment of selected patients with PFIC3 caused by mutations in the ATP-binding sites of ABCB4. Full article

(This article belongs to the Special Issue Structure-Based Understanding of the Function-Dysfunction of ABC Transporters)

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14 pages, 3166 KiB

Open AccessArticle

Acting on the CFTR Membrane-Spanning Domains Interface Rescues Some Misfolded Mutants

by Nesrine Baatallah, Ahmad Elbahnsi, Benoit Chevalier, Solène Castanier, Jean-Paul Mornon, Iwona Pranke, Aleksander Edelman, Isabelle Sermet-Gaudelus, Isabelle Callebaut and Alexandre Hinzpeter

Int. J. Mol. Sci. 2022, 23(24), 16225; https://doi.org/10.3390/ijms232416225 - 19 Dec 2022

Cited by 1 | Viewed by 1665

Abstract

ABC transporters are large membrane proteins sharing a complex architecture, which comprises two nucleotide-binding domains (NBDs) and two membrane-spanning domains (MSDs). These domains are susceptible to mutations affecting their folding and assembly. In the CFTR (ABCC7) protein, a groove has been highlighted in [...] Read more.

ABC transporters are large membrane proteins sharing a complex architecture, which comprises two nucleotide-binding domains (NBDs) and two membrane-spanning domains (MSDs). These domains are susceptible to mutations affecting their folding and assembly. In the CFTR (ABCC7) protein, a groove has been highlighted in the MSD1 at the level of the membrane inner leaflet, containing both multiple mutations affecting folding and a binding site for pharmaco-chaperones that stabilize this region. This groove is also present in ABCB proteins, however it is covered by a short elbow helix, while in ABCC proteins it remains unprotected, due to a lower position of the elbow helix in the presence of the ABCC-specific lasso motif. Here, we identified a MSD1 second-site mutation located in the vicinity of the CFTR MSD1 groove that partially rescued the folding defect of cystic fibrosis causing mutations located within MSD1, while having no effect on the most frequent mutation, F508del, located within NBD1. A model of the mutated protein 3D structure suggests additional interaction between MSD1 and MSD2, strengthening the assembly at the level of the MSD intracellular loops. Altogether, these results provide insightful information in understanding key features of the folding and function of the CFTR protein in particular, and more generally, of type IV ABC transporters. Full article

(This article belongs to the Special Issue Structure-Based Understanding of the Function-Dysfunction of ABC Transporters)

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16 pages, 2908 KiB

Open AccessArticle

5-Arylidenerhodanines as P-gp Modulators: An Interesting Effect of the Carboxyl Group on ABCB1 Function in Multidrug-Resistant Cancer Cells

by Ewa Żesławska, Waldemar Tejchman, Annamária Kincses, Gabriella Spengler, Wojciech Nitek, Grzegorz Żuchowski and Ewa Szymańska

Int. J. Mol. Sci. 2022, 23(18), 10812; https://doi.org/10.3390/ijms231810812 - 16 Sep 2022

Cited by 4 | Viewed by 1335

Abstract

Multidrug resistance (MDR) is considered one of the major mechanisms responsible for the failure of numerous anticancer and antiviral chemotherapies. Various strategies to overcome the MDR phenomenon have been developed, and one of the most attractive research directions is focused on the inhibition [...] Read more.

Multidrug resistance (MDR) is considered one of the major mechanisms responsible for the failure of numerous anticancer and antiviral chemotherapies. Various strategies to overcome the MDR phenomenon have been developed, and one of the most attractive research directions is focused on the inhibition of MDR transporters, membrane proteins that extrude cytotoxic drugs from living cells. Here, we report the results of our studies on a series newly synthesized of 5-arylidenerhodanines and their ability to inhibit the ABCB1 efflux pump in mouse T-lymphoma cancer cells. In the series, compounds possessing a triphenylamine moiety and the carboxyl group in their structure were of particular interest. These amphiphilic compounds showed over 17-fold stronger efflux pump inhibitory effects than verapamil. The cytotoxic and antiproliferative effects of target rhodanines on T-lymphoma cells were also investigated. A putative binding mode for 11, one of the most potent P-gp inhibitors tested here, was predicted by molecular docking studies and discussed with regard to the binding mode of verapamil. Full article

(This article belongs to the Special Issue Structure-Based Understanding of the Function-Dysfunction of ABC Transporters)

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14 pages, 30214 KiB

Open AccessArticle

In Vitro Rescue of the Bile Acid Transport Function of ABCB11 Variants by CFTR Potentiators

by Elodie Mareux, Martine Lapalus, Amel Ben Saad, Renaud Zelli, Mounia Lakli, Yosra Riahi, Marion Almes, Manon Banet, Isabelle Callebaut, Jean-Luc Decout, Thomas Falguières, Emmanuel Jacquemin and Emmanuel Gonzales

Int. J. Mol. Sci. 2022, 23(18), 10758; https://doi.org/10.3390/ijms231810758 - 15 Sep 2022

Cited by 2 | Viewed by 1720

Abstract

ABCB11 is responsible for biliary bile acid secretion at the canalicular membrane of hepatocytes. Variations in the ABCB11 gene cause a spectrum of rare liver diseases. The most severe form is progressive familial intrahepatic cholestasis type 2 (PFIC2). Current medical treatments have limited [...] Read more.

ABCB11 is responsible for biliary bile acid secretion at the canalicular membrane of hepatocytes. Variations in the ABCB11 gene cause a spectrum of rare liver diseases. The most severe form is progressive familial intrahepatic cholestasis type 2 (PFIC2). Current medical treatments have limited efficacy. Here, we report the in vitro study of Abcb11 missense variants identified in PFIC2 patients and their functional rescue using cystic fibrosis transmembrane conductance regulator potentiators. Three ABCB11 disease-causing variations identified in PFIC2 patients (i.e., A257V, T463I and G562D) were reproduced in a plasmid encoding an Abcb11-green fluorescent protein. After transfection, the expression and localization of the variants were studied in HepG2 cells. Taurocholate transport activity and the effect of potentiators were studied in Madin–Darby canine kidney (MDCK) clones coexpressing Abcb11 and the sodium taurocholate cotransporting polypeptide (Ntcp/Slc10A1). As predicted using three-dimensional structure analysis, the three variants were expressed at the canalicular membrane but showed a defective function. Ivacaftor, GLP1837, SBC040 and SBC219 potentiators increased the bile acid transport of A257V and T463I and to a lesser extent, of G562D Abcb11 missense variants. In addition, a synergic effect was observed when ivacaftor was combined with SBC040 or SBC219. Such potentiators could represent new pharmacological approaches for improving the condition of patients with ABCB11 deficiency due to missense variations affecting the function of the transporter. Full article

(This article belongs to the Special Issue Structure-Based Understanding of the Function-Dysfunction of ABC Transporters)

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16 pages, 2699 KiB

Open AccessArticle

Comprehensive Collection and Prediction of ABC Transmembrane Protein Structures in the AI Era of Structural Biology

by Hedvig Tordai, Erzsebet Suhajda, Ian Sillitoe, Sreenath Nair, Mihaly Varadi and Tamas Hegedus

Int. J. Mol. Sci. 2022, 23(16), 8877; https://doi.org/10.3390/ijms23168877 - 09 Aug 2022

Cited by 7 | Viewed by 2723

Abstract

The number of unique transmembrane (TM) protein structures doubled in the last four years, which can be attributed to the revolution of cryo-electron microscopy. In addition, AlphaFold2 (AF2) also provided a large number of predicted structures with high quality. However, if a specific [...] Read more.

The number of unique transmembrane (TM) protein structures doubled in the last four years, which can be attributed to the revolution of cryo-electron microscopy. In addition, AlphaFold2 (AF2) also provided a large number of predicted structures with high quality. However, if a specific protein family is the subject of a study, collecting the structures of the family members is highly challenging in spite of existing general and protein domain-specific databases. Here, we demonstrate this and assess the applicability and usability of automatic collection and presentation of protein structures via the ABC protein superfamily. Our pipeline identifies and classifies transmembrane ABC protein structures using the PFAM search and also aims to determine their conformational states based on special geometric measures, conftors. Since the AlphaFold database contains structure predictions only for single polypeptide chains, we performed AF2-Multimer predictions for human ABC half transporters functioning as dimers. Our AF2 predictions warn of possibly ambiguous interpretation of some biochemical data regarding interaction partners and call for further experiments and experimental structure determination. We made our predicted ABC protein structures available through a web application, and we joined the 3D-Beacons Network to reach the broader scientific community through platforms such as PDBe-KB. Full article

(This article belongs to the Special Issue Structure-Based Understanding of the Function-Dysfunction of ABC Transporters)

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Review

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13 pages, 3103 KiB

Open AccessReview

Snapshots of ABCG1 and ABCG5/G8: A Sterol’s Journey to Cross the Cellular Membranes

by Fatemeh Rezaei, Danny Farhat, Gonca Gursu, Sabrina Samnani and Jyh-Yeuan Lee

Int. J. Mol. Sci. 2023, 24(1), 484; https://doi.org/10.3390/ijms24010484 - 28 Dec 2022

Cited by 9 | Viewed by 1932

Abstract

The subfamily-G ATP-binding cassette (ABCG) transporters play important roles in regulating cholesterol homeostasis. Recent progress in the structural data of ABCG1 and ABCG5/G8 disclose putative sterol binding sites that suggest the possible cholesterol translocation pathway. ABCG1 and ABCG5/G8 share high similarity in the [...] Read more.

The subfamily-G ATP-binding cassette (ABCG) transporters play important roles in regulating cholesterol homeostasis. Recent progress in the structural data of ABCG1 and ABCG5/G8 disclose putative sterol binding sites that suggest the possible cholesterol translocation pathway. ABCG1 and ABCG5/G8 share high similarity in the overall molecular architecture, and both transporters appear to use several unique structural motifs to facilitate cholesterol transport along this pathway, including the phenylalanine highway and the hydrophobic valve. Interestingly, ABCG5/G8 is known to transport cholesterol and phytosterols, whereas ABCG1 seems to exclusively transport cholesterol. Ligand docking analysis indeed suggests a difference in recruiting sterol molecules to the known sterol-binding sites. Here, we further discuss how the different and shared structural features are relevant to their physiological functions, and finally provide our perspective on future studies in ABCG cholesterol transporters. Full article

(This article belongs to the Special Issue Structure-Based Understanding of the Function-Dysfunction of ABC Transporters)

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