10 results on '"Braakman, Ineke"'
Search Results
2. Intramolecular quality control: HIV-1 envelope gp160 signal-peptide cleavage as a functional folding checkpoint
- Author
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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, Sub Cellular Protein Chemistry, Cellular Protein Chemistry, Medical Microbiology and Infection Prevention, and AII - Infectious diseases
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Signal peptide ,disulfide isomerization ,envelope glycoprotein ,membrane tethering ,HIV Envelope Protein gp120 ,Protein Sorting Signals ,Virus Replication ,Cleavage (embryo) ,Biochemistry ,General Biochemistry, Genetics and Molecular Biology ,HIV Envelope Protein gp160 ,Structure-Activity Relationship ,chemistry.chemical_compound ,03 medical and health sciences ,Protein structure ,Biosynthesis ,protein folding ,Humans ,Protein Interaction Domains and Motifs ,signal peptide ,Cysteine ,030304 developmental biology ,0303 health sciences ,Protein Stability ,Chemistry ,Biochemistry, Genetics and Molecular Biology(all) ,Endoplasmic reticulum ,C-terminus ,030302 biochemistry & molecular biology ,Viral Load ,redox-active cysteine ,Cell biology ,3. Good health ,Folding (chemistry) ,N-terminus ,gp120 ,endoplasmic reticulum ,HEK293 Cells ,Secretory protein ,Intramolecular force ,Biophysics ,HIV-1 ,Protein folding ,disulfide bond ,Protein Processing, Post-Translational ,HeLa Cells ,Genetics and Molecular Biology(all) - Abstract
SummaryRemoval of the membrane-tethering signal peptides that target secretory proteins to the endoplasmic reticulum is a prerequisite for proper folding. While generally thought to be removed well before translation termination, we here report two novel post-targeting functions for the HIV-1 gp120 signal peptide, which remains attached until gp120 folding triggers its removal. First, the signal peptide improves fidelity of folding by enhancing conformational plasticity of gp120 by driving disulfide isomerization through a redox-active cysteine, at the same time delaying folding by tethering the N-terminus to the membrane, which needs assembly with the C-terminus. Second, its carefully timed cleavage represents intramolecular quality control and ensures release and stabilization of (only) natively folded gp120. Postponed cleavage and the redox-active cysteine both are highly conserved and important for viral fitness. Considering the ∼15% secretory proteins in our genome and the frequency of N-to-C contacts in protein structures, these regulatory roles of the signal peptide are bound to be more common in secretory-protein biosynthesis.
- Published
- 2021
3. Characterization of CNPY5 and its family members
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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
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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
4. The CFTR P67L variant reveals a key role for N-terminal lasso helices in channel folding, maturation, and pharmacologic rescue
- Author
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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, Sub Cellular Protein Chemistry, and Cellular Protein Chemistry
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Protein Conformation, alpha-Helical ,0301 basic medicine ,Protein Folding ,Protein Conformation ,Mutant ,Cystic Fibrosis Transmembrane Conductance Regulator ,Biology ,Molecular Dynamics Simulation ,medicine.disease_cause ,Biochemistry ,Cell Line ,03 medical and health sciences ,chemistry.chemical_compound ,Protein structure ,medicine ,Missense mutation ,Humans ,Molecular Biology ,Mutation ,030102 biochemistry & molecular biology ,HEK 293 cells ,Lumacaftor ,alpha-Helical ,Cell Biology ,Cystic Fibrosis Transmembrane Conductance Regulator/chemistry ,Cystic fibrosis transmembrane conductance regulator ,Cell biology ,030104 developmental biology ,chemistry ,biology.protein ,Protein folding - 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
- Published
- 2021
5. Analysis of Disulfide Bond Formation
- Author
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Braakman, Ineke, Lamriben, Lydia, van Zadelhoff, Guus, Hebert, Daniel N., Sub Cellular Protein Chemistry, Cellular Protein Chemistry, Sub Cellular Protein Chemistry, and Cellular Protein Chemistry
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0301 basic medicine ,radiolabeling ,Lysis ,Immunoprecipitation ,disulfide bonds ,Endosomes ,Sulfur Radioisotopes ,Biochemistry ,Article ,pulse-chase ,03 medical and health sciences ,Methionine ,Structural Biology ,protein folding ,Animals ,Humans ,Cysteine ,Disulfides ,Protein disulfide-isomerase ,Polyacrylamide gel electrophoresis ,chemistry.chemical_classification ,030102 biochemistry & molecular biology ,Staining and Labeling ,Endoplasmic reticulum ,Amino acid ,secretory pathway ,endoplasmic reticulum ,030104 developmental biology ,HEK293 Cells ,chemistry ,Ethylmaleimide ,Protein Biosynthesis ,Microsome ,Protein folding ,Electrophoresis, Polyacrylamide Gel ,Oxidation-Reduction - Abstract
In this unit, protocols are provided for detection of disulfide bond formation in cultures of intact cells and in an in vitro translation system containing isolated microsomes or semi-permeabilized cells. First, the newly synthesized protein of interest is biosynthetically labeled with radioactive amino acids in a short pulse. The labeled protein then is chased with unlabeled amino acids. At different times during the chase, a sample is collected, membranes are lysed with detergent, and the protein is isolated by immunoprecipitation, as described. A support protocol is provided for analysis of disulfide bonds in the immunoprecipitates by SDS-PAGE with and without prior reduction. The difference in mobility observed between the gels with nonreduced and reduced samples is due to disulfide bonds in the nonreduced protein. An additional support protocol is included that uses PEG-maleimide to modify free thiols and follow disulfide-bond formation by SDS-PAGE. © 2017 by John Wiley & Sons, Inc.
- Published
- 2017
6. Alteration of protein function by a silent polymorphism linked to tRNA abundance
- Author
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Kirchner, Sebastian, Cai, Zhiwei, Rauscher, Robert, Kastelic, Nicolai, Anding, Melanie, Czech, Andreas, Kleizen, Bertrand, Ostedgaard, Lynda S., Braakman, Ineke, Sheppard, David N., Ignatova, Zoya, Sub Cellular Protein Chemistry, Cellular Protein Chemistry, Sub Cellular Protein Chemistry, and Cellular Protein Chemistry
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0301 basic medicine ,Cystic Fibrosis ,Pulmonology ,Cultured tumor cells ,Gene Expression ,Cystic Fibrosis Transmembrane Conductance Regulator ,Ribosome ,Biochemistry ,0302 clinical medicine ,Protein structure ,Single Channel Recording ,RNA, Transfer ,Medicine and Health Sciences ,Biology (General) ,Membrane Electrophysiology ,Genetics ,Protein Stability ,General Neuroscience ,Messenger RNA ,Translation (biology) ,Cystic fibrosis transmembrane conductance regulator ,Nucleic acids ,Bioassays and Physiological Analysis ,Genetic Diseases ,Transfer RNA ,Cell lines ,Cellular Structures and Organelles ,General Agricultural and Biological Sciences ,Biological cultures ,Research Article ,Silent mutation ,QH301-705.5 ,Biology ,Research and Analysis Methods ,Transfection ,Polymorphism, Single Nucleotide ,General Biochemistry, Genetics and Molecular Biology ,03 medical and health sciences ,Structure-Activity Relationship ,Autosomal Recessive Diseases ,Humans ,HeLa cells ,Non-coding RNA ,Molecular Biology Techniques ,Molecular Biology ,Institut für Biochemie und Biologie ,Silent Mutation ,Clinical Genetics ,General Immunology and Microbiology ,Biology and life sciences ,Electrophysiological Techniques ,RNA ,Cell Biology ,Cell cultures ,Fibrosis ,030104 developmental biology ,HEK293 Cells ,biology.protein ,Protein Translation ,Ribosomes ,030217 neurology & neurosurgery ,Developmental Biology - Abstract
Synonymous single nucleotide polymorphisms (sSNPs) are considered neutral for protein function, as by definition they exchange only codons, not amino acids. We identified an sSNP that modifies the local translation speed of the cystic fibrosis transmembrane conductance regulator (CFTR), leading to detrimental changes to protein stability and function. This sSNP introduces a codon pairing to a low-abundance tRNA that is particularly rare in human bronchial epithelia, but not in other human tissues, suggesting tissue-specific effects of this sSNP. Up-regulation of the tRNA cognate to the mutated codon counteracts the effects of the sSNP and rescues protein conformation and function. Our results highlight the wide-ranging impact of sSNPs, which invert the programmed local speed of mRNA translation and provide direct evidence for the central role of cellular tRNA levels in mediating the actions of sSNPs in a tissue-specific manner., Author summary Synonymous single nucleotide polymorphisms (sSNPs) occur at high frequency in the human genome and are associated with ~50 diseases in humans; the responsible molecular mechanisms remain enigmatic. Here, we investigate the impact of the common sSNP, T2562G, on cystic fibrosis transmembrane conductance regulator (CFTR). Although this sSNP, by itself, does not cause cystic fibrosis (CF), it is prevalent in patients with CFTR-related disorders. T2562G sSNP modifies the local translation speed at the Thr854 codon, leading to changes in CFTR stability and channel function. This sSNP introduces a codon pairing to a low-abundance tRNA, which is particularly rare in human bronchial epithelia, but not in other human tissues, suggesting a tissue-specific effect of this sSNP. Enhancement of the cellular concentration of the tRNA cognate to the mutant ACG codon rescues the stability and conduction defects of T2562G-CFTR. These findings reveal an unanticipated mechanism—inverting the programmed local speed of mRNA translation in a tRNA-dependent manner—for sSNP-associated diseases.
- Published
- 2017
7. Folding of influenza virus hemagglutinin in insect cells is fast and efficient
- Author
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Li, Xin, van Oers, Monique M, Vlak, Just M, Braakman, Ineke, Sub Cellular Protein Chemistry, Cellular Protein Chemistry, Sub Cellular Protein Chemistry, and Cellular Protein Chemistry
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Protein Folding ,ph ,Laboratory of Virology ,Hemagglutinins, Viral ,Hemagglutinin (influenza) ,Bioengineering ,Sf9 ,Context (language use) ,calnexin ,Spodoptera ,Applied Microbiology and Biotechnology ,Virus ,Cell Line ,expression system ,oligomerization ,Laboratorium voor Virologie ,quality-control ,Calnexin ,vaccine ,membrane glycoprotein ,Animals ,Humans ,biology ,Endoplasmic reticulum ,disulfide bond formation ,endoplasmic-reticulum ,General Medicine ,Orthomyxoviridae ,PE&RC ,Folding (chemistry) ,Biochemistry ,biology.protein ,escherichia-coli ,Protein folding ,HeLa Cells ,Biotechnology - Abstract
Folding of influenza virus hemagglutinin (HA) in the endoplasmic reticulum has been well defined in mammalian cells. In different mammalian cell lines the protein follows the same folding pathway with identical folding intermediates, but folds with very different kinetics. To examine the effect of cellular context on HA folding and to test to which extent insect cells would support the HA folding process, we expressed HA in Sf9 insect cells. Strikingly, in this invertebrate system HA folded faster and more efficiently, still via the same folding intermediates as in vertebrate cells. Our results suggest that insect cells provide a highly efficient and effective folding environment for influenza virus HA and the ideal production platform for HA (emergency) vaccines.
- Published
- 2015
8. Deletion of the Highly Conserved N-Glycan at Asn260 of HIV-1 gp120 Affects Folding and Lysosomal Degradation of gp120, and Results in Loss of Viral Infectivity.
- Author
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Mathys, Leen, François, Katrien O., Quandte, Matthias, Braakman, Ineke, and Balzarini, Jan
- Subjects
GLYCANS ,LYSOSOMAL storage diseases ,GLYCOPROTEINS ,BIOSYNTHESIS ,METHIONINE ,CD4 antigen - Abstract
N-linked glycans covering the surface of the HIV-1 glycoprotein gp120 are of major importance for the correct folding of this glycoprotein. Of the, on average, 24 N-linked glycans present on gp120, the glycan at Asn260 was reported to be essential for the correct expression of gp120 and gp41 in the virus particle and deletion of the N260 glycan in gp120 heavily compromised virus infectivity. We show here that gp160 containing the N260Q mutation reaches the Golgi apparatus during biosynthesis. Using pulse-chase experiments with [
35 S] methionine/cysteine, we show that oxidative folding was slightly delayed in case of mutant N260Q gp160 and that CD4 binding was markedly compromised compared to wild-type gp160. In the search of compensatory mutations, we found a mutation in the V1/V2 loop of gp120 (S128N) that could partially restore the infectivity of mutant N260Q gp120 virus. However, the mutation S128N did not enhance any of the above-mentioned processes so its underlying compensatory mechanism must be a conformational effect that does not affect CD4 binding per se. Finally, we show that mutant N260Q gp160 was cleaved to gp120 and gp41 to a much lower extent than wild-type gp160, and that it was subject of lysosomal degradation to a higher extent than wild-type gp160 showing a prominent role of this process in the breakdown of N260-glycan-deleted gp160, which could not be counteracted by the S128N mutation. Moreover, at least part of the wild-type or mutant gp160 that is normally targeted for lysosomal degradation reached a conformation that enabled CD4 binding. [ABSTRACT FROM AUTHOR]- Published
- 2014
- Full Text
- View/download PDF
9. The return of the peroxisome.
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Van Der Zand, Adabella, Braakman, Ineke, Geuze, Hans J., and Tabak, Henk F.
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PEROXISOMES , *CELLS , *EUKARYOTIC cells , *METABOLISM , *PHYSIOLOGY , *BIOCHEMISTRY , *FATTY acids , *BIOMOLECULES - Abstract
Of the classical compartments of eukaryotic cells, peroxisomes were the last to be discovered. They are small, single-membrane-bound vesicles involved in cellular metabolism, most notably the β-oxidation of fatty acids. Characterization of their properties and behavior has progressed rather slowly. However, during the past few years, peroxisomes have entered the limelight as a result of several breakthroughs. These include the observations that they are not autonomously multiplying organelles but are derived from the endoplasmic reticulum, and that partitioning of peroxisomes to progeny cells is an active and well-controlled process. In addition, we are discovering more and more proteins that are not only dedicated to peroxisomes but also serve other organelles. [ABSTRACT FROM AUTHOR]
- Published
- 2006
- Full Text
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10. Slowing ribosome velocity restores folding and function of mutant CFTR.
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Oliver, Kathryn E., Rauscher, Robert, Mijnders, Marjolein, Wei Wang, Wolpert, Matthew J., Maya, Jessica, Sabusap, Carleen M., Kesterson, Robert A., Kirk, Kevin L., Rab, Andras, Braakman, Ineke, Jeong S. Hong, Hartman IV, John L., Ignatova, Zoya, Sorscher, Eric J., Wang, Wei, Hong, Jeong S, and Hartman, John L 4th
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VELOCITY , *RIBOSOMAL proteins , *PROTEIN synthesis , *CYSTIC fibrosis , *COLON (Anatomy) , *PROTEIN metabolism , *AMINOPYRIDINES , *ANIMAL experimentation , *BIOCHEMISTRY , *BRONCHI , *COMPARATIVE studies , *CYTOLOGY , *CYTOPLASM , *EPITHELIAL cells , *EPITHELIUM , *GENES , *HETEROCYCLIC compounds , *ILEUM , *PHENOMENOLOGY , *RESEARCH methodology , *MEDICAL cooperation , *MEMBRANE proteins , *MICE , *MOLECULAR structure , *GENETIC mutation , *PANCREAS , *PROTEINS , *RATS , *RESEARCH , *EVALUATION research , *INDOLE compounds - 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. [ABSTRACT FROM AUTHOR]
- Published
- 2019
- Full Text
- View/download PDF
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