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5. Slowing ribosome velocity restores folding and function of mutant CFTR

Author

Oliver, Kathryn E., Rauscher, Robert, Mijnders, Marjolein, Wang, Wei, Wolpert, Matthew J., Maya, Jessica, Sabusap, Carleen M., Kesterson, Robert A., Kirk, Kevin L., Rab, Andras, Braakman, Ineke, Hong, Jeong S., Hartman, John L., IV, Ignatova, Zoya, and Sorscher, Eric J.

Subjects
Thermo Fisher Scientific Inc., Cystic fibrosis, Tezacaftor, Translation (Genetics), Lumacaftor, Scientific equipment industry, Protein synthesis, Criminal investigation, Biosynthesis, Fibrosis, Health care industry, The University of Alabama at Birmingham
Abstract

Cystic fibrosis (CF) is caused by mutations in the CF transmembrane conductance regulator (CFTR), with approximately 90% of patients harboring at least one copy of the disease-associated variant F508del. We utilized a yeast phenomic system to identify genetic modifiers of F508del-CFTR biogenesis, from which ribosomal protein L12 (RPL12/uL11) emerged as a molecular target. In the present study, we investigated mechanism(s) by which suppression of RPL12 rescues F508del protein synthesis and activity. Using ribosome profiling, we found that rates of translation initiation and elongation were markedly slowed by RPL12 silencing. However, proteolytic stability and patch-clamp assays revealed RPL12 depletion significantly increased F508del-CFTR steady-state expression, interdomain assembly, and baseline open-channel probability. We next evaluated whether Rpl12-corrected F508del-CFTR could be further enhanced with concomitant pharmacologic repair (e.g., using clinically approved modulators lumacaftor and tezacaftor) and demonstrated additivity of these treatments. Rpl12 knockdown also partially restored maturation of specific CFTR variants in addition to F508del, and WT Cftr biogenesis was enhanced in the pancreas, colon, and ileum of Rpl12 haplosufficient mice. Modulation of ribosome velocity therefore represents a robust method for understanding both CF pathogenesis and therapeutic response., Introduction Cystic fibrosis (CF) is caused by abnormalities of the CF transmembrane conductance regulator (CFTR), with the most common variant being F508del (deletion of phenylalanine at position 508). The F508del [...]

Published
2019
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8. Two phases of disulfide bond formation have differing requirements for oxygen

Author

Koritzinsky, Marianne, Levitin, Fiana, van den Beucken, Twan, Rumantir, Ryan A, Harding, Nicholas J, Chu, Kenneth C, Boutros, Paul C, Braakman, Ineke, and Wouters, Bradly G

Subjects
Generic health relevance, Cell Hypoxia, Disulfides, Endoplasmic Reticulum, HCT116 Cells, HeLa Cells, Humans, Isomerism, Models, Biological, Oxygen, Protein Folding, Protein Processing, Post-Translational, RNA, Messenger, Transcription, Genetic, Hela Cells, Biological Sciences, Medical and Health Sciences, Developmental Biology
Abstract

Most proteins destined for the extracellular space require disulfide bonds for folding and stability. Disulfide bonds are introduced co- and post-translationally in endoplasmic reticulum (ER) cargo in a redox relay that requires a terminal electron acceptor. Oxygen can serve as the electron acceptor in vitro, but its role in vivo remains unknown. Hypoxia causes ER stress, suggesting a role for oxygen in protein folding. Here we demonstrate the existence of two phases of disulfide bond formation in living mammalian cells, with differential requirements for oxygen. Disulfide bonds introduced rapidly during protein synthesis can occur without oxygen, whereas those introduced during post-translational folding or isomerization are oxygen dependent. Other protein maturation processes in the secretory pathway, including ER-localized N-linked glycosylation, glycan trimming, Golgi-localized complex glycosylation, and protein transport, occur independently of oxygen availability. These results suggest that an alternative electron acceptor is available transiently during an initial phase of disulfide bond formation and that post-translational oxygen-dependent disulfide bond formation causes hypoxia-induced ER stress.

Published
2013

12. Neil J. Bulleid (1960-2023), a virtuoso of protein folding and redox biology

Author

Sub Cellular Protein Chemistry, Cellular Protein Chemistry, Braakman, Ineke, High, Stephen, Kadler, Karl, Sitia, Roberto, Tokatlidis, Kostas, Woodman, Philip, Sub Cellular Protein Chemistry, Cellular Protein Chemistry, Braakman, Ineke, High, Stephen, Kadler, Karl, Sitia, Roberto, Tokatlidis, Kostas, and Woodman, Philip

Published
2023

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

Author

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

Published
2023

14. ABC-transporter CFTR folds with high fidelity through a modular, stepwise pathway

Author

Sub Cellular Protein Chemistry, Cellular Protein Chemistry, Im, Jisu, Hillenaar, Tamara, Yeoh, Hui Ying, Sahasrabudhe, Priyanka, Mijnders, Marjolein, van Willigen, Marcel, Hagos, Azib, de Mattos, Eduardo, van der Sluijs, Peter, Braakman, Ineke, Sub Cellular Protein Chemistry, Cellular Protein Chemistry, Im, Jisu, Hillenaar, Tamara, Yeoh, Hui Ying, Sahasrabudhe, Priyanka, Mijnders, Marjolein, van Willigen, Marcel, Hagos, Azib, de Mattos, Eduardo, van der Sluijs, Peter, and Braakman, Ineke

Published
2023

15. Structure basis of CFTR folding, function and pharmacology

Author

Sub Cellular Protein Chemistry, Cellular Protein Chemistry, Hwang, Tzyh-Chang, Braakman, Ineke, van der Sluijs, Peter, Callebaut, Isabelle, Sub Cellular Protein Chemistry, Cellular Protein Chemistry, Hwang, Tzyh-Chang, Braakman, Ineke, van der Sluijs, Peter, and Callebaut, Isabelle

Published
2023

20. Redefining Hypo- and Hyper-Responding Phenotypes of CFTR Mutants for Understanding and Therapy

Author

Hillenaar, Tamara, Beekman, Jeffrey, van der Sluijs, Peter, Braakman, Ineke, Sub Cellular Protein Chemistry, Cellular Protein Chemistry, Sub Cellular Protein Chemistry, and Cellular Protein Chemistry

Subjects
Organic Chemistry, hyper-responders, hypo-responding mutants, General Medicine, Catalysis, Computer Science Applications, Inorganic Chemistry, protein folding, modulators, CFTR, Physical and Theoretical Chemistry, Molecular Biology, Spectroscopy
Abstract

Mutations in CFTR cause misfolding and decreased or absent ion-channel function, resulting in the disease Cystic Fibrosis. Fortunately, a triple-modulator combination therapy (Trikafta) has been FDA approved for 178 mutations, including all patients who have F508del on one allele. That so many CFTR mutants respond well to modulators developed for a single mutation is due to the nature of the folding process of this multidomain protein. We have addressed the question ‘What characterizes the exceptions: the mutants that functionally respond either not or extremely well’.A functional response is the product of the number of CFTR molecules on the cell surface, open probability, and conductivity of the CFTR chloride channel. By combining biosynthetic radiolabeling with protease-susceptibility assays, we have followed CF-causing mutants during early and late stages of folding in presence and absence of modulators. Most CFTR mutants showed typical biochemical responses for each modulator, such as a TMD1 conformational change or an increase of (cell-surface) stability, regardless of a functional response. These modulators thus should still be considered for hypo-responder genotypes. Understanding both biochemical and functional phenotypes of outlier mutations will boost our insights into CFTR folding and misfolding, and lead to improved therapeutic strategies.

Published
2022

25. The importance of naturally attenuated SARS-CoV-2in the fight against COVID-19

Author

Armengaud, Jean [0000-0003-1589-445X], Thuret, Jean-Yves [0000-0001-5385-7620], Anken, Eelco van [0000-0001-9529-2701], Acosta-Alvear, Diego [0000-0002-1139-8486], Aragón, Tomás [0000-0002-1700-2729], Arias, Carolina [0000-0002-4445-0826], Blondel, Marc [0000-0003-4897-2995], Braakman, Ineke [0000-0003-1592-4364], Collet, Jean-François [0000-0001-8069-7036], Courcol, René [0000-0003-2324-5687], Danchin, Antoine [0000-0002-6350-5001], Deleuze, Jean-François [0000-0002-5358-4463], Lavigne, Jean-Philippe [0000-0002-9484-0304], Lucas, Sophie [0000-0003-1287-7996], Michiels, Thomas [0000-0001-9615-8053], Moore, Edward R.B. [0000-0001-7693-924X], Nixon-Abell, Jonathon [0000-0003-4169-0012], Rosselló-Mora, Ramón [0000-0001-8253-3107], Shi, Zheng-Li [0000-0001-8089-163X], Siccardi, Antonio G. [0000-0002-1654-5545], Sitia, Roberto [0000-0001-7086-4152], Tillett, Daniel [0000-0003-1061-0489], Timmis, Kenneth N. [0000-0002-0066-4670], Toledano, Michel B. [0000-0002-3079-1179], Sluijs, Peter van der [0000-0002-4485-3342], Vicenzi, Elisa [0000-0003-0051-3968], Armengaud, Jean, Delaunay, Agnes, Thuret, Jean-Yves, Anken, Eelco van, Acosta-Alvear, Diego, Aragón, Tomás, Arias, Carolina, Blondel, Marc, Braakman, Ineke, Collet, Jean-François, Courcol, René, Danchin, Antoine, Deleuze, Jean-François, Lavigne, Jean-Philippe, Lucas, Sophie, Michiels, Thomas, Moore, Edward R.B., Nixon-Abell, Jonathon, Rosselló-Mora, Ramón, Shi, Zheng-Li, Siccardi, Antonio G., Sitia, Roberto, Tillett, Daniel, Timmis, Kenneth N., Toledano, Michel B., Sluijs, Peter van der, Vicenzi, Elisa, Armengaud, Jean [0000-0003-1589-445X], Thuret, Jean-Yves [0000-0001-5385-7620], Anken, Eelco van [0000-0001-9529-2701], Acosta-Alvear, Diego [0000-0002-1139-8486], Aragón, Tomás [0000-0002-1700-2729], Arias, Carolina [0000-0002-4445-0826], Blondel, Marc [0000-0003-4897-2995], Braakman, Ineke [0000-0003-1592-4364], Collet, Jean-François [0000-0001-8069-7036], Courcol, René [0000-0003-2324-5687], Danchin, Antoine [0000-0002-6350-5001], Deleuze, Jean-François [0000-0002-5358-4463], Lavigne, Jean-Philippe [0000-0002-9484-0304], Lucas, Sophie [0000-0003-1287-7996], Michiels, Thomas [0000-0001-9615-8053], Moore, Edward R.B. [0000-0001-7693-924X], Nixon-Abell, Jonathon [0000-0003-4169-0012], Rosselló-Mora, Ramón [0000-0001-8253-3107], Shi, Zheng-Li [0000-0001-8089-163X], Siccardi, Antonio G. [0000-0002-1654-5545], Sitia, Roberto [0000-0001-7086-4152], Tillett, Daniel [0000-0003-1061-0489], Timmis, Kenneth N. [0000-0002-0066-4670], Toledano, Michel B. [0000-0002-3079-1179], Sluijs, Peter van der [0000-0002-4485-3342], Vicenzi, Elisa [0000-0003-0051-3968], Armengaud, Jean, Delaunay, Agnes, Thuret, Jean-Yves, Anken, Eelco van, Acosta-Alvear, Diego, Aragón, Tomás, Arias, Carolina, Blondel, Marc, Braakman, Ineke, Collet, Jean-François, Courcol, René, Danchin, Antoine, Deleuze, Jean-François, Lavigne, Jean-Philippe, Lucas, Sophie, Michiels, Thomas, Moore, Edward R.B., Nixon-Abell, Jonathon, Rosselló-Mora, Ramón, Shi, Zheng-Li, Siccardi, Antonio G., Sitia, Roberto, Tillett, Daniel, Timmis, Kenneth N., Toledano, Michel B., Sluijs, Peter van der, and Vicenzi, Elisa

Abstract

The current SARS‐CoV‐2 pandemic is wreaking havoc throughout the world and has rapidly become a global health emergency. A central question concerning COVID‐19 is why some individuals become sick and others not. Many have pointed already at variation in risk factors between individuals. However, the variable outcome of SARS‐CoV‐2 infections may, at least in part, be due also to differences between the viral subspecies with which individuals are infected. A more pertinent question is how we are to overcome the current pandemic. A vaccine against SARS‐CoV‐2 would offer significant relief, although vaccine developers have warned that design, testing and production of vaccines may take a year if not longer. Vaccines are based on a handful of different designs (i), but the earliest vaccines were based on the live, attenuated virus. As has been the case for other viruses during earlier pandemics, SARS‐CoV‐2 will mutate and may naturally attenuate over time (ii). What makes the current pandemic unique is that, thanks to state‐of‐the‐art nucleic acid sequencing technologies, we can follow in detail how SARS‐CoV‐2 evolves while it spreads. We argue that knowledge of naturally emerging attenuated SARS‐CoV‐2 variants across the globe should be of key interest in our fight against the pandemic.

Published
2020

29. The importance of naturally attenuated SARS‐CoV ‐2 in the fight against COVID ‐19

Author

Armengaud, Jean, Delaunay‐Moisan, Agnès, Thuret, Jean‐Yves, Anken, Eelco, Acosta‐Alvear, Diego, Aragón, Tomás, Arias, Carolina, Blondel, Marc, Braakman, Ineke, Collet, Jean‐François, Courcol, René, Danchin, Antoine, Deleuze, Jean‐François, Lavigne, Jean‐Philippe, Lucas, Sophie, Michiels, Thomas, Moore, Edward R. B., Nixon‐Abell, Jonathon, Rossello‐Mora, Ramon, Shi, Zheng‐Li, Siccardi, Antonio G., Sitia, Roberto, Tillett, Daniel, Timmis, Kenneth N., Toledano, Michel B., Sluijs, Peter, Vicenzi, Elisa, Médicaments et Technologies pour la Santé (MTS), Université Paris-Saclay-Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Institut National de Recherche pour l’Agriculture, l’Alimentation et l’Environnement (INRAE), Institut de Biologie Intégrative de la Cellule (I2BC), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Paris-Saclay-Centre National de la Recherche Scientifique (CNRS), Stress Oxydatif et Cancer (SOC), Département Biologie Cellulaire (BioCell), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Paris-Saclay-Centre National de la Recherche Scientifique (CNRS)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Paris-Saclay-Centre National de la Recherche Scientifique (CNRS)-Institut de Biologie Intégrative de la Cellule (I2BC), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Paris-Saclay-Centre National de la Recherche Scientifique (CNRS)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Paris-Saclay-Centre National de la Recherche Scientifique (CNRS), Sénescence et stabilité génomique (SEN), Département Biologie des Génomes (DBG), San Raffaele Scientific Institute, Vita-Salute San Raffaele University and Center for Translational Genomics and Bioinformatics, University of California [Santa Barbara] (UC Santa Barbara), University of California (UC), Universidad Pública de Navarra [Espagne] = Public University of Navarra (UPNA), Génétique, génomique fonctionnelle et biotechnologies (UMR 1078) (GGB), EFS-Université de Brest (UBO)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Institut Brestois Santé Agro Matière (IBSAM), Université de Brest (UBO), Université de Bretagne Occidentale - UFR Médecine et Sciences de la Santé (UBO UFR MSS), Utrecht University [Utrecht], Université Catholique de Louvain = Catholic University of Louvain (UCL), Walloon Excellence in Life sciences and BIOtechnology [Liège] (WELBIO), Institut Cochin (IC UM3 (UMR 8104 / U1016)), Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)-Université Paris Cité (UPCité), Centre National de Recherche en Génomique Humaine (CNRGH), Commissariat à l'énergie atomique et aux énergies alternatives (CEA), Institut de Biologie François JACOB (JACOB), Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA), Virulence bactérienne et maladies infectieuses (VBMI), Institut National de la Santé et de la Recherche Médicale (INSERM)-Université de Montpellier (UM), University of Gothenburg (GU), Sahlgrenska University Hospital [Gothenburg], University of Cambridge [UK] (CAM), Institut Mediterrani d'Estudis Avancats (IMEDEA), Consejo Superior de Investigaciones Científicas [Madrid] (CSIC)-Universidad de las Islas Baleares (UIB), Wuhan Institute of Virology [Wuhan, China], Chinese Academy of Sciences [Wuhan Branch], Technische Universität Braunschweig = Technical University of Braunschweig [Braunschweig], ANR-17-CE18-0023,Phylopeptidomics,Identification rapide de bactéries pathogènes et résistances aux antibiotiques(2017), Armengaud, Jean [0000-0003-1589-445X], Thuret, Jean-Yves [0000-0001-5385-7620], Anken, Eelco van [0000-0001-9529-2701], Acosta-Alvear, Diego [0000-0002-1139-8486], Aragón, Tomás [0000-0002-1700-2729], Arias, Carolina [0000-0002-4445-0826], Blondel, Marc [0000-0003-4897-2995], Braakman, Ineke [0000-0003-1592-4364], Collet, Jean-François [0000-0001-8069-7036], Courcol, René [0000-0003-2324-5687], Danchin, Antoine [0000-0002-6350-5001], Deleuze, Jean-François [0000-0002-5358-4463], Lavigne, Jean-Philippe [0000-0002-9484-0304], Lucas, Sophie [0000-0003-1287-7996], Michiels, Thomas [0000-0001-9615-8053], Moore, Edward R.B. [0000-0001-7693-924X], Nixon-Abell, Jonathon [0000-0003-4169-0012], Rosselló-Mora, Ramón [0000-0001-8253-3107], Shi, Zheng-Li [0000-0001-8089-163X], Siccardi, Antonio G. [0000-0002-1654-5545], Sitia, Roberto [0000-0001-7086-4152], Tillett, Daniel [0000-0003-1061-0489], Timmis, Kenneth N. [0000-0002-0066-4670], Toledano, Michel B. [0000-0002-3079-1179], Sluijs, Peter van der [0000-0002-4485-3342], Vicenzi, Elisa [0000-0003-0051-3968], University of California [Santa Barbara] (UCSB), University of California, Institut Brestois Santé Agro Matière (IBSAM), Université de Brest (UBO)-Université de Brest (UBO)-EFS-Institut National de la Santé et de la Recherche Médicale (INSERM), Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)-Université de Paris (UP), Technical University Braunschweig, Armengaud, Jean, Thuret, Jean-Yves, Anken, Eelco van, Acosta-Alvear, Diego, Aragón, Tomás, Arias, Carolina, Blondel, Marc, Braakman, Ineke, Collet, Jean-François, Courcol, René, Danchin, Antoine, Deleuze, Jean-François, Lavigne, Jean-Philippe, Lucas, Sophie, Michiels, Thomas, Moore, Edward R.B., Nixon-Abell, Jonathon, Rosselló-Mora, Ramón, Shi, Zheng-Li, Siccardi, Antonio G., Sitia, Roberto, Tillett, Daniel, Timmis, Kenneth N., Toledano, Michel B., Sluijs, Peter van der, Vicenzi, Elisa, Armengaud, J., Delaunay-Moisan, A., Thuret, J. -Y., van Anken, E., Acosta-Alvear, D., Aragon, T., Arias, C., Blondel, M., Braakman, I., Collet, J. -F., Courcol, R., Danchin, A., Deleuze, J. -F., Lavigne, J. -P., Lucas, S., Michiels, T., Moore, E. R. B., Nixon-Abell, J., Rossello-Mora, R., Shi, Z., Siccardi, A. G., Sitia, R., Tillett, D., Timmis, K. N., Toledano, M. B., van der Sluijs, P., Vicenzi, E., and UCL - SSS/DDUV - Institut de Duve

Subjects
Opinion, viruses, Pneumonia, Viral, Gene Expression, Microbiology, Disease Outbreaks, Evolution, Molecular, Betacoronavirus, [SDV.MHEP.MI]Life Sciences [q-bio]/Human health and pathology/Infectious diseases, Animals, Humans, Health emergency, Selection, Genetic, Pandemics, Ecology, Evolution, Behavior and Systematics, [SDV.MHEP.ME]Life Sciences [q-bio]/Human health and pathology/Emerging diseases, Virulence, SARS-CoV-2, fungi, COVID-19, SARS Virus, Adaptation, Physiological, Severe acute respiratory syndrome-related coronavirus, Host-Pathogen Interactions, Mutation, Spike Glycoprotein, Coronavirus, [SDV.MP.VIR]Life Sciences [q-bio]/Microbiology and Parasitology/Virology, Coronavirus Infections
Abstract

The current SARS‐CoV‐2 pandemic is wreaking havoc throughout the world and has rapidly become a global health emergency. A central question concerning COVID‐19 is why some individuals become sick and others not. Many have pointed already at variation in risk factors between individuals. However, the variable outcome of SARS‐CoV‐2 infections may, at least in part, be due also to differences between the viral subspecies with which individuals are infected. A more pertinent question is how we are to overcome the current pandemic. A vaccine against SARS‐CoV‐2 would offer significant relief, although vaccine developers have warned that design, testing and production of vaccines may take a year if not longer. Vaccines are based on a handful of different designs (i), but the earliest vaccines were based on the live, attenuated virus. As has been the case for other viruses during earlier pandemics, SARS‐CoV‐2 will mutate and may naturally attenuate over time (ii). What makes the current pandemic unique is that, thanks to state‐of‐the‐art nucleic acid sequencing technologies, we can follow in detail how SARS‐CoV‐2 evolves while it spreads. We argue that knowledge of naturally emerging attenuated SARS‐CoV‐2 variants across the globe should be of key interest in our fight against the pandemic.

Published
2020
Full Text
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36. Characterization of CNPY5 and its family members

Author

Schildknegt, Danny, Lodder, Naomi, Pandey, Abhinav, Egmond, Maarten, Pena, Florentina, Braakman, Ineke, van der Sluijs, Peter, Sub Cellular Protein Chemistry, Sub Membrane Biochemistry & Biophysics, Cellular Protein Chemistry, Membrane Biochemistry and Biophysics, Sub Cellular Protein Chemistry, Sub Membrane Biochemistry & Biophysics, Cellular Protein Chemistry, and Membrane Biochemistry and Biophysics

Subjects
Full‐Length Papers, pERp1, Biochemistry, Dithiothreitol, 03 medical and health sciences, chemistry.chemical_compound, MZB1, Growth factor receptor, Full‐Length Paper, protein folding, CNPY5, Cell adhesion, Receptor, Molecular Biology, Canopy (CNPY) proteins, 030304 developmental biology, 0303 health sciences, B lymphocyte, Chemistry, Endoplasmic reticulum, 030302 biochemistry & molecular biology, Cell biology, Secretory protein, ER, Protein folding, Cysteine
Abstract

The Canopy (CNPY) family consists of four members predicted to be soluble proteins localized to the endoplasmic reticulum (ER). They are involved in a wide array of processes, including angiogenesis, cell adhesion, and host defense. CNPYs are thought to do so via regulation of secretory transport of a diverse group of proteins, such as immunoglobulin M, growth factor receptors, toll‐like receptors, and the low‐density lipoprotein receptor. Thus far, a comparative analysis of the mammalian CNPY family is missing. Bioinformatic analysis shows that mammalian CNPYs, except the CNPY1 homolog, have N‐terminal signal sequences and C‐terminal ER‐retention signals and that mammals have an additional member CNPY5, also known as plasma cell‐induced ER protein 1/marginal zone B cell‐specific protein 1. Canopy proteins are particularly homologous in four hydrophobic alpha‐helical regions and contain three conserved disulfide bonds. This sequence signature is characteristic for the saposin‐like superfamily and strongly argues that CNPYs share this common saposin fold. We showed that CNPY2, 3, 4, and 5 (termed CNPYs) localize to the ER. In radioactive pulse‐chase experiments, we found that CNPYs rapidly form disulfide bonds and fold within minutes into their native forms. Disulfide bonds in native CNPYs remain sensitive to low concentrations of dithiothreitol (DTT) suggesting that the cysteine residues forming them are relatively accessible to solutes. Possible roles of CNPYs in the folding of secretory proteins in the ER are discussed.

Published
2019

40. Co-Translational Folding of the First Transmembrane Domain of ABC-Transporter CFTR is Supported by Assembly with the First Cytosolic Domain

Author

Kleizen, Bertrand, primary, van Willigen, Marcel, additional, Mijnders, Marjolein, additional, Peters, Florence, additional, Grudniewska, Magda, additional, Hillenaar, Tamara, additional, Thomas, Ann, additional, Kooijman, Laurens, additional, Peters, Kathryn W., additional, Frizzell, Raymond, additional, van der Sluijs, Peter, additional, and Braakman, Ineke, additional

Published
2021
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41. The CFTR P67L variant reveals a key role for N-terminal lasso helices in channel folding, maturation, and pharmacologic rescue

Author

Sub Cellular Protein Chemistry, Cellular Protein Chemistry, Sabusap, Carleen Mae, Joshi, Disha, Simhaev, Luba, Oliver, Kathryn E., Senderowitz, Hanoch, van Willigen, Marcel, Braakman, Ineke, Rab, Andras, Sorscher, Eric J., Hong, Jeong S., Sub Cellular Protein Chemistry, Cellular Protein Chemistry, Sabusap, Carleen Mae, Joshi, Disha, Simhaev, Luba, Oliver, Kathryn E., Senderowitz, Hanoch, van Willigen, Marcel, Braakman, Ineke, Rab, Andras, Sorscher, Eric J., and Hong, Jeong S.

Published
2021

42. Intramolecular quality control: HIV-1 envelope gp160 signal-peptide cleavage as a functional folding checkpoint

Author

Sub Cellular Protein Chemistry, Cellular Protein Chemistry, McCaul, Nicholas, Quandte, Matthias, Bontjer, Ilja, van Zadelhoff, Guus, Land, Aafke, Crooks, Ema T., Binley, James M., Sanders, Rogier W., Braakman, Ineke, Sub Cellular Protein Chemistry, Cellular Protein Chemistry, McCaul, Nicholas, Quandte, Matthias, Bontjer, Ilja, van Zadelhoff, Guus, Land, Aafke, Crooks, Ema T., Binley, James M., Sanders, Rogier W., and Braakman, Ineke

Published
2021

43. Co-Translational Folding of the First Transmembrane Domain of ABC-Transporter CFTR is Supported by Assembly with the First Cytosolic Domain

Author

Sub Cellular Protein Chemistry, Sub Onderwijsbeleid, Cellular Protein Chemistry, Kleizen, Bertrand, van Willigen, Marcel, Mijnders, Marjolein, Peters, Floor, Grudniewska, Magda, Hillenaar, Tamara, Thomas, Ann, Kooijman, Laurens, Peters, Kathryn W., Frizzell, Raymond, van der Sluijs, Peter, Braakman, Ineke, Sub Cellular Protein Chemistry, Sub Onderwijsbeleid, Cellular Protein Chemistry, Kleizen, Bertrand, van Willigen, Marcel, Mijnders, Marjolein, Peters, Floor, Grudniewska, Magda, Hillenaar, Tamara, Thomas, Ann, Kooijman, Laurens, Peters, Kathryn W., Frizzell, Raymond, van der Sluijs, Peter, and Braakman, Ineke

Published
2021

44. Clinical and molecular characterization of the R751L-CFTR mutation

Author

Sub Cellular Protein Chemistry, Cellular Protein Chemistry, Haq, Iram J., Althaus, Mike, Gardner, Aaron Ions, Yeoh, Hui Ying, Joshi, Urjita, Saint-Criq, Vinciane, Verdon, Bernard, Townshend, Jennifer, O'Brien, Christopher, Ben-Hamida, Mahfud, Thomas, Matthew, Bourke, Stephen, van der Sluijs, Peter, Braakman, Ineke, Ward, Chris, Gray, Michael A., Brodlie, Malcolm, Sub Cellular Protein Chemistry, Cellular Protein Chemistry, Haq, Iram J., Althaus, Mike, Gardner, Aaron Ions, Yeoh, Hui Ying, Joshi, Urjita, Saint-Criq, Vinciane, Verdon, Bernard, Townshend, Jennifer, O'Brien, Christopher, Ben-Hamida, Mahfud, Thomas, Matthew, Bourke, Stephen, van der Sluijs, Peter, Braakman, Ineke, Ward, Chris, Gray, Michael A., and Brodlie, Malcolm

Published
2021

45. Clinical and molecular characterization of the R751L-CFTR mutation

Author

Haq, Iram J., Althaus, Mike, Gardner, Aaron Ions, Yeoh, Hui Ying, Joshi, Urjita, Saint-Criq, Vinciane, Verdon, Bernard, Townshend, Jennifer, O'Brien, Christopher, Ben-Hamida, Mahfud, Thomas, Matthew, Bourke, Stephen, van der Sluijs, Peter, Braakman, Ineke, Ward, Chris, Gray, Michael A., Brodlie, Malcolm, Haq, Iram J., Althaus, Mike, Gardner, Aaron Ions, Yeoh, Hui Ying, Joshi, Urjita, Saint-Criq, Vinciane, Verdon, Bernard, Townshend, Jennifer, O'Brien, Christopher, Ben-Hamida, Mahfud, Thomas, Matthew, Bourke, Stephen, van der Sluijs, Peter, Braakman, Ineke, Ward, Chris, Gray, Michael A., and Brodlie, Malcolm

Abstract

Cysticfibrosis (CF) arises from mutations in the CF transmembrane conductance regulator (CFTR) gene, resulting in progressiveand life-limiting respiratory disease. R751L is a rare CFTR mutation that is poorly characterized. Our aims were to describe theclinical and molecular phenotypes associated with R751L. Relevant clinical data were collected from three heterozygote individu-als harboring R751L (2 patients with G551D/R751L and 1 with F508del/R751L). Assessment of R751L-CFTR function was made inprimary human bronchial epithelial cultures (HBEs) andXenopusoocytes. Molecular properties of R751L-CFTR were investigatedin the presence of known CFTR modulators. Although sweat chloride was elevated in all three patients, the clinical phenotypeassociated with R751L was mild. Chloride secretion in F508del/R751L HBEs was reduced compared with non-CF HBEs and asso-ciated with a reduction in sodium absorption by the epithelial sodium channel (ENaC). However, R751L-CFTR function inXenopusoocytes, together with folding and cell surface transport of R751L-CFTR, was not different from wild-type CFTR. Overall,R751L-CFTR was associated with reduced sodium chloride absorption but had functional properties similar to wild-type CFTR.This is thefirst report of R751L-CFTR that combines clinical phenotype with characterization of functional and biological proper-ties of the mutant channel. Our work will build upon existing knowledge of mutations within this region of CFTR and, importantly,inform approaches for clinical management. Elevated sweat chloride and reduced chloride secretion in HBEs may be due to al-ternative non-CFTR factors, which require further investigation.

Published
2021

46. Co-Translational Folding of the First Transmembrane Domain of ABC-Transporter CFTR is Supported by Assembly with the First Cytosolic Domain

Author

MDL onderzoek 1, Kleizen, Bertrand, van Willigen, Marcel, Mijnders, Marjolein, Peters, Florence, Grudniewska, Magda, Hillenaar, Tamara, Thomas, Ann, Kooijman, Laurens, Peters, Kathryn W, Frizzell, Raymond, van der Sluijs, Peter, Braakman, Ineke, MDL onderzoek 1, Kleizen, Bertrand, van Willigen, Marcel, Mijnders, Marjolein, Peters, Florence, Grudniewska, Magda, Hillenaar, Tamara, Thomas, Ann, Kooijman, Laurens, Peters, Kathryn W, Frizzell, Raymond, van der Sluijs, Peter, and Braakman, Ineke

Published
2021

47. Co-Translational Folding of the First Transmembrane Domain of ABC-Transporter CFTR is Supported by Assembly with the First Cytosolic Domain

Author

Kleizen, Bertrand, van Willigen, Marcel, Mijnders, Marjolein, Peters, Floor, Grudniewska, Magda, Hillenaar, Tamara, Thomas, Ann, Kooijman, Laurens, Peters, Kathryn W., Frizzell, Raymond, van der Sluijs, Peter, Braakman, Ineke, Sub Cellular Protein Chemistry, Sub Onderwijsbeleid, and Cellular Protein Chemistry

Subjects
Models, Molecular, Protein Folding, Mutant, domain assembly, Aminopyridines, Cystic Fibrosis Transmembrane Conductance Regulator, ATP-binding cassette transporter, Protein Structure, Secondary, Evolution, Molecular, 03 medical and health sciences, 0302 clinical medicine, helical packing, Cytosol, Protein Domains, Structural Biology, Humans, Benzodioxoles, Genes, Suppressor, Molecular Biology, 030304 developmental biology, 0303 health sciences, Chemistry, Translation (biology), multispanning membrane proteins, Transmembrane protein, Cell biology, Folding (chemistry), Transmembrane domain, co-translational, HEK293 Cells, Protein Biosynthesis, Protein folding, 030217 neurology & neurosurgery, Function (biology), HeLa Cells, Peptide Hydrolases
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.

Published
2020

48. Efficient IgM assembly and secretion require the plasma cell induced endoplasmic reticulum protein pERp1

Author

van Anken, Eelco, Pena, Florentina, Hafkemeijer, Nicole, Christis, Chantal, Romijn, Edwin P., Oorschot, Ulla Grauschop Viola M.J., Pertel, Thomas, Engels, Sander, Ora, Ari, Lastun, Viorica, Glockshuber, Rudi, Klumperman, Judith, Heck, Albert J.R., Luban, Jeremy, and Braakman, Ineke

Subjects
Immunoglobulins -- Research, Endoplasmic reticulum -- Research, Protein folding -- Research, Science and technology
Abstract

Plasma cells daily secrete their own mass in antibodies, which fold and assemble in the endoplasmic reticulum (ER). To reach these levels, cells require pERp1, a novel lymphocyte-specific small ER-resident protein, which attains expression levels as high as BiP when B cells differentiate into plasma cells. Although pERp1 has no homology with known ER proteins, it does contain a CXXC motif typical for oxidoreductases. In steady state, the CXXC cysteines are locked by two parallel disulfide bonds with a downstream C[(X).sub.6]C motif, and pERp1 displays only modest oxidoreductase activity, pERpl emerged as a dedicated folding factor for IgM, associating with both heavy and light chains and promoting assembly and secretion of mature IgM.

Published
2009

49. Clinical and molecular characterization of the R751L-CFTR mutation

Author

Haq, Iram J., primary, Althaus, Mike, additional, Gardner, Aaron Ions, additional, Yeoh, Hui Ying, additional, Joshi, Urjita, additional, Saint-Criq, Vinciane, additional, Verdon, Bernard, additional, Townshend, Jennifer, additional, O’Brien, Christopher, additional, Ben-Hamida, Mahfud, additional, Thomas, Matthew, additional, Bourke, Stephen, additional, van der Sluijs, Peter, additional, Braakman, Ineke, additional, Ward, Chris, additional, Gray, Michael A., additional, and Brodlie, Malcolm, additional

Published
2021
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50. Exchange between ordered and disordered segments in CFTR modulates function at the expense of stability: A molecular pathway for misfolding of CFTR

Author

Govaerts, Cédric, Goormaghtigh, Erik, Braakman, Ineke, Hendrix, Jelle, Prévost, Martine, Garcia-Pino, Abel, Tompa, Peter, Scholl, Daniel, Govaerts, Cédric, Goormaghtigh, Erik, Braakman, Ineke, Hendrix, Jelle, Prévost, Martine, Garcia-Pino, Abel, Tompa, Peter, and Scholl, Daniel

Abstract

The genetic disease cystic fibrosis is the most common lethal genetic disease in Western countries. People born with cystic fibrosis suffer from many health issues including severe respiratory problems, inflammation and recurrent lung infections that can become fatal. The disease is caused by the loss of function of a protein called the cystic fibrosis transmembrane conductance regulator (CFTR). CFTR is an chloride ion channel and, in healthy people, its activity assures correct water and salt transport across the cell membrane. Most cases of cystic fibrosis are caused by a genetic defect that leads to the deletion of phenylalanine 508 (F508del) in the amino acid sequence of the protein. The molecular mechanism by which F508del leads to loss of function of the CFTR channel is still poorly understood. The mutation is found in the first nucleotide binding domain (NBD1) and studies have shown that it causes misfolding of CFTR and subsequent degradation of the protein by the cellular quality control system. It is established that the mutation affects stability and dynamics of NBD1 but does not alter its structure significantly. This destabilizing effect of F508del can be compensated by specific mutations distributed over different regions of NBD1, leading to recovery of membrane expression of a functional channel. A surprising example involves the regulatory insertion (RI), a 32-residue long segment found in all CFTR orthologs but not in related channels or transporters. The RI is not resolved in crystal structures of NBD1 nor cryo-EM structures of CFTR and has been described as intrinsically disordered. Its functional role in CFTR is unknown. Removal of the RI increases the stability of the NBD1 domain and, in the context of F508del-CFTR, this deletion restores maturation, cell surface expression and activity of the mutant channel. We probed the effect of the RI on NBD1 structure, dynamics and allostery using X-ray crystallography, single molecule FRET and hydrogen-deu, Doctorat en Sciences, info:eu-repo/semantics/nonPublished

Published
2020

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