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1.
Nat Commun ; 14(1): 7823, 2023 Nov 28.
Article in English | MEDLINE | ID: mdl-38016954

ABSTRACT

Ultra-tight binding is usually observed for proteins associating with rigidified molecules. Previously, we demonstrated that femtomolar binders derived from the Armadillo repeat proteins (ArmRPs) can be designed to interact very tightly with fully flexible peptides. Here we show for ArmRPs with four and seven sequence-identical internal repeats that the peptide-ArmRP complexes display conformational dynamics. These dynamics stem from transient breakages of individual protein-residue contacts that are unrelated to overall unbinding. The labile contacts involve electrostatic interactions. We speculate that these dynamics allow attaining very high binding affinities, since they reduce entropic losses. Importantly, only NMR techniques can pick up these local events by directly detecting conformational exchange processes without complications from changes in solvent entropy. Furthermore, we demonstrate that the interaction surface of the repeat protein regularizes upon peptide binding to become more compatible with the peptide geometry. These results provide novel design principles for ultra-tight binders.


Subject(s)
Carrier Proteins , Peptides , Carrier Proteins/metabolism , Peptides/chemistry , Proteins/metabolism , Armadillo Domain Proteins/metabolism , Entropy , Protein Binding , Protein Conformation
2.
Biochemistry ; 62(2): 318-329, 2023 01 17.
Article in English | MEDLINE | ID: mdl-35657362

ABSTRACT

High protein stability is an important feature for proteins used as therapeutics, as diagnostics, and in basic research. We have previously employed consensus design to engineer optimized Armadillo repeat proteins (ArmRPs) for sequence-specific recognition of linear epitopes with a modular binding mode. These designed ArmRPs (dArmRPs) feature high stability and are composed of M-type internal repeats that are flanked by N- and C-terminal capping repeats that protect the hydrophobic core from solvent exposure. While the overall stability of the designed ArmRPs is remarkably high, subsequent biochemical and biophysical experiments revealed that the N-capping repeat assumes a partially unfolded, solvent-accessible conformation for a small fraction of time that renders it vulnerable to proteolysis and aggregation. To overcome this problem, we have designed new N-caps starting from an M-type internal repeat using the Rosetta software. The superior stability of the computationally refined models was experimentally verified by circular dichroism and nuclear magnetic resonance spectroscopy. A crystal structure of a dArmRP containing the novel N-cap revealed that the enhanced stability correlates with an improved packing of this N-cap onto the hydrophobic core of the dArmRP. Hydrogen exchange experiments further show that the level of local unfolding of the N-cap is reduced by several orders of magnitude, resulting in increased resistance to proteolysis and weakened aggregation. As a first application of the novel N-cap, we determined the solution structure of a dArmRP with four internal repeats, which was previously impeded by the instability of the original N-cap.


Subject(s)
Armadillo Domain Proteins , Protein Conformation , Models, Molecular , Armadillo Domain Proteins/chemistry , Magnetic Resonance Spectroscopy , Protein Stability
3.
Nat Commun ; 12(1): 4696, 2021 08 04.
Article in English | MEDLINE | ID: mdl-34349113

ABSTRACT

Productive ribosomal RNA (rRNA) compaction during ribosome assembly necessitates establishing correct tertiary contacts between distant secondary structure elements. Here, we quantify the response of the yeast proteome to low temperature (LT), a condition where aberrant mis-paired RNA folding intermediates accumulate. We show that, at LT, yeast cells globally boost production of their ribosome assembly machinery. We find that the LT-induced assembly factor, Puf6, binds to the nascent catalytic RNA-rich subunit interface within the 60S pre-ribosome, at a site that eventually loads the nuclear export apparatus. Ensemble Förster resonance energy transfer studies show that Puf6 mimics the role of Mg2+ to usher a unique long-range tertiary contact to compact rRNA. At LT, puf6 mutants accumulate 60S pre-ribosomes in the nucleus, thus unveiling Puf6-mediated rRNA compaction as a critical temperature-regulated rescue mechanism that counters rRNA misfolding to prime export competence.


Subject(s)
Cell Nucleus/metabolism , RNA-Binding Proteins/metabolism , Ribosome Subunits, Large, Eukaryotic/metabolism , Saccharomyces cerevisiae Proteins/metabolism , Active Transport, Cell Nucleus , Cold Temperature , GTP Phosphohydrolases/metabolism , Mutation , Protein Binding , Protein Interaction Domains and Motifs , Proteome/metabolism , RNA Folding , RNA Precursors/chemistry , RNA Precursors/metabolism , RNA, Ribosomal/chemistry , RNA, Ribosomal/metabolism , RNA-Binding Proteins/chemistry , RNA-Binding Proteins/genetics , Ribosome Subunits, Large, Eukaryotic/chemistry , Ribosomes/metabolism , Saccharomyces cerevisiae/genetics , Saccharomyces cerevisiae/metabolism , Saccharomyces cerevisiae/physiology , Saccharomyces cerevisiae Proteins/chemistry , Saccharomyces cerevisiae Proteins/genetics
4.
Chimia (Aarau) ; 75(6): 505-507, 2021 Jun 30.
Article in English | MEDLINE | ID: mdl-34233813

ABSTRACT

Heteronuclear NMR in combination with isotope labelling is used to study folding of polypeptides induced by metals in the case of metallothioneins, binding of the peptidic allosteric modulator ρ-TIA to the human G-protein coupled α1b adrenergic receptor, the development of therapeutic drugs that interfere with the biosynthesis of the outer membrane of Gram-negative bacteria, and a system in which protein assembly is induced upon peptide addition. NMR in these cases is used to derive precise structural data and to study the dynamics.


Subject(s)
Peptides , Receptors, Adrenergic, alpha-1 , Humans
5.
Cell Death Differ ; 26(10): 2157-2178, 2019 Oct.
Article in English | MEDLINE | ID: mdl-30728462

ABSTRACT

Post-transcriptional control of mRNAs by RNA-binding proteins (RBPs) has a prominent role in the regulation of gene expression. RBPs interact with mRNAs to control their biogenesis, splicing, transport, localization, translation, and stability. Defects in such regulation can lead to a wide range of human diseases from neurological disorders to cancer. Many RBPs are conserved between Caenorhabditis elegans and humans, and several are known to regulate apoptosis in the adult C. elegans germ line. How these RBPs control apoptosis is, however, largely unknown. Here, we identify mina-1(C41G7.3) in a RNA interference-based screen as a novel regulator of apoptosis, which is exclusively expressed in the adult germ line. The absence of MINA-1 causes a dramatic increase in germ cell apoptosis, a reduction in brood size, and an impaired P granules organization and structure. In vivo crosslinking immunoprecipitation experiments revealed that MINA-1 binds a set of mRNAs coding for RBPs associated with germ cell development. Additionally, a system-wide analysis of a mina-1 deletion mutant compared with wild type, including quantitative proteome and transcriptome data, hints to a post-transcriptional regulatory RBP network driven by MINA-1 during germ cell development in C. elegans. In particular, we found that the germline-specific Argonaute WAGO-4 protein levels are increased in mina-1 mutant background. Phenotypic analysis of double mutant mina-1;wago-4 revealed that contemporary loss of MINA-1 and WAGO-4 strongly rescues the phenotypes observed in mina-1 mutant background. To strengthen this functional interaction, we found that upregulation of WAGO-4 in mina-1 mutant animals causes hypersensitivity to exogenous RNAi. Our comprehensive experimental approach allowed us to describe a phenocritical interaction between two RBPs controlling germ cell apoptosis and exogenous RNAi. These findings broaden our understanding of how RBPs can orchestrate different cellular events such as differentiation and death in C. elegans.


Subject(s)
Argonaute Proteins/genetics , Argonaute Proteins/metabolism , Caenorhabditis elegans Proteins/genetics , Caenorhabditis elegans Proteins/metabolism , Caenorhabditis elegans/genetics , Caenorhabditis elegans/metabolism , RNA Interference , Animals , Germ Cells
6.
Biol Chem ; 400(3): 395-404, 2019 02 25.
Article in English | MEDLINE | ID: mdl-30517075

ABSTRACT

Designed armadillo repeat proteins (dArmRPs) are modular peptide binders composed of N- and C-terminal capping repeats Y and A and a variable number of internal modules M that each specifically recognize two amino acids of the target peptide. Complementary fragments of dArmRPs obtained by splitting the protein between helices H1 and H2 of an internal module show conditional and specific assembly only in the presence of a target peptide (Michel, E., Plückthun, A., and Zerbe, O. (2018). Peptide-guided assembly of repeat protein fragments. Angew. Chem. Int. Ed. 57, 4576-4579). Here, we investigate dArmRP fragments that already spontaneously assemble with high affinity, e.g. those obtained from splits between entire modules or between helices H2 and H3. We find that the interaction of the peptide with the assembled fragments induces distal conformational rearrangements that suggest an induced fit on a global protein level. A population analysis of an equimolar mixture of an N-terminal and three C-terminal fragments with various affinities for the target peptide revealed predominant assembly of the weakest peptide binder. However, adding a target peptide to this mixture altered the population of the protein complexes such that the combination with the highest affinity for the peptide increased and becomes predominant when adding excess of peptide, highlighting the feasibility of peptide-induced enrichment of best binders from inter-modular fragment mixtures.


Subject(s)
Armadillo Domain Proteins/chemistry , Peptides/chemistry , Models, Molecular , Nuclear Magnetic Resonance, Biomolecular , Protein Binding
7.
Nat Commun ; 9(1): 3669, 2018 09 10.
Article in English | MEDLINE | ID: mdl-30201955

ABSTRACT

Disordered extensions at the termini and short internal insertions distinguish eukaryotic ribosomal proteins (r-proteins) from their anucleated archaeal counterparts. Here, we report an NMR structure of such a eukaryotic-specific segment (ESS) in the r-protein eS26 in complex with the escortin Tsr2. The structure reveals how ESS attracts Tsr2 specifically to importin:eS26 complexes entering the nucleus in order to trigger non-canonical RanGTP-independent disassembly. Tsr2 then sequesters the released eS26 and prevents rebinding to the importin, providing an alternative allosteric mechanism to terminate the process of nuclear import. Notably, a Diamond-Blackfan anemia-associated Tsr2 mutant protein is impaired in binding to ESS, unveiling a critical role for this interaction in human hematopoiesis. We propose that eS26-ESS and Tsr2 are components of a nuclear sorting system that co-evolved with the emergence of the nucleocytoplasmic barrier and transport carriers.


Subject(s)
Apoptosis Regulatory Proteins/metabolism , Karyopherins/metabolism , Ribosomal Proteins/metabolism , Active Transport, Cell Nucleus , Allosteric Site , Cell Nucleus/metabolism , Circular Dichroism , Cytoplasm/metabolism , Hematopoiesis , Humans , In Situ Hybridization, Fluorescence , Magnetic Resonance Spectroscopy , Mass Spectrometry , Mutation , Nuclear Proteins/metabolism , Phenotype , Protein Binding , Protein Conformation , RNA/chemistry , Recombinant Proteins/metabolism , Ribosomes/metabolism , Saccharomyces cerevisiae , ran GTP-Binding Protein/metabolism
8.
Methods Mol Biol ; 1737: 177-195, 2018.
Article in English | MEDLINE | ID: mdl-29484594

ABSTRACT

Posttranscriptional regulation of gene expression by small noncoding RNAs (sRNAs) is an important control mechanism that modulates bacterial metabolism, motility, and pathogenesis. Using the bacterial carbon storage regulator/regulator of secondary metabolism (Csr/Rsm) system, we here describe an E. coli-based cell-free translation assay that allows a quantitative analysis of translation regulation by ncRNAs and their corresponding translation repressor proteins. The assay quantifies the translation of chloramphenicol acetyltransferase in cell-free expression reactions that contain defined amounts of ncRNA and repressor protein. We demonstrate our protocol with a comparative translation activation analysis of the RsmX, RsmY, and RsmZ sRNAs from Pseudomonas protegens, which reveals a superior efficacy of RsmZ over RsmX and RsmY.


Subject(s)
Bacterial Proteins/metabolism , Gene Expression Regulation, Bacterial , Protein Biosynthesis , Pseudomonas/genetics , RNA, Bacterial/genetics , RNA, Small Untranslated/genetics , RNA-Binding Proteins/metabolism , Bacterial Proteins/genetics , Pseudomonas/growth & development , RNA-Binding Proteins/genetics
9.
Angew Chem Int Ed Engl ; 57(17): 4576-4579, 2018 04 16.
Article in English | MEDLINE | ID: mdl-29480529

ABSTRACT

Herein, we present the peptide-guided assembly of complementary fragments of designed armadillo repeat proteins (dArmRPs) to create proteins that bind peptides not only with high affinity but also with good selectivity. We recently demonstrated that complementary N- and C-terminal fragments of dArmRPs form high-affinity complexes that resemble the structure of the full-length protein, and that these complexes bind their target peptides. We now demonstrate that dArmRPs can be split such that the fragments assemble only in the presence of a templating peptide, and that fragment mixtures enrich the combination with the highest affinity for this peptide. The enriched fragment combination discriminates single amino acid variations in the target peptide with high specificity. Our results suggest novel opportunities for the generation of new peptide binders by selection from dArmRP fragment mixtures.


Subject(s)
Peptides/chemistry , Proteins/chemical synthesis , Models, Molecular , Proteins/chemistry
10.
Methods Enzymol ; 565: 389-422, 2015.
Article in English | MEDLINE | ID: mdl-26577740

ABSTRACT

The steady technical advances of nuclear magnetic resonance (NMR) over the past decades enabled a significant increase in the molecular size of protein particles that can be subjected to a structural and functional characterization in solution. The larger molecular weight of such proteins is accompanied with an increase in NMR signals that complicate spectral interpretation due to signal overlap. The application of segmental isotope labeling to selected domains in multi-domain proteins can significantly facilitate spectral interpretation by reducing the number of observable signals. However, severe signal overlap may persist within individual domains that show low signal dispersion. To further reduce the number of signals and spectral complexity in such systems, we developed a procedure for selective amino acid-type labeling in individual domains of multi-domain proteins. This strategy combines efficient amino acid-type labeling amenable by cell-free protein expression with near-seamless domain ligation achievable by expressed protein ligation. By application of simple dual labeling schemes, this approach further allows residue-specific isotope labeling to position NMR-observable probes at desired sites within segments of multi-domain proteins. This chapter describes a detailed protocol for selective amino acid-type segmental labeling of multi-domain proteins and illustrates its application to a multi-domain RNA-binding protein. The applied ligation approach is further suitable for efficient ligation of unlabeled and/or uniformly labeled domains produced solely by recombinant in vivo expression.


Subject(s)
Amino Acids/metabolism , Proteins/metabolism , Isotope Labeling , Magnetic Resonance Spectroscopy
11.
Nature ; 509(7502): 588-92, 2014 May 29.
Article in English | MEDLINE | ID: mdl-24828038

ABSTRACT

MicroRNA and protein sequestration by non-coding RNAs (ncRNAs) has recently generated much interest. In the bacterial Csr/Rsm system, which is considered to be the most general global post-transcriptional regulatory system responsible for bacterial virulence, ncRNAs such as CsrB or RsmZ activate translation initiation by sequestering homodimeric CsrA-type proteins from the ribosome-binding site of a subset of messenger RNAs. However, the mechanism of ncRNA-mediated protein sequestration is not understood at the molecular level. Here we show for Pseudomonas fluorescens that RsmE protein dimers assemble sequentially, specifically and cooperatively onto the ncRNA RsmZ within a narrow affinity range. This assembly yields two different native ribonucleoprotein structures. Using a powerful combination of nuclear magnetic resonance and electron paramagnetic resonance spectroscopy we elucidate these 70-kilodalton solution structures, thereby revealing the molecular mechanism of the sequestration process and how RsmE binding protects the ncRNA from RNase E degradation. Overall, our findings suggest that RsmZ is well-tuned to sequester, store and release RsmE and therefore can be viewed as an ideal protein 'sponge'.


Subject(s)
Escherichia coli Proteins/metabolism , Methyltransferases/metabolism , Protein Binding , RNA, Untranslated/metabolism , Animals , Base Sequence , Electron Spin Resonance Spectroscopy , Escherichia coli/chemistry , Escherichia coli/genetics , Escherichia coli/metabolism , Escherichia coli Proteins/chemistry , Methyltransferases/chemistry , Models, Biological , Models, Molecular , Molecular Weight , Nuclear Magnetic Resonance, Biomolecular , Nucleic Acid Conformation , Protein Multimerization , RNA, Untranslated/chemistry , RNA, Untranslated/genetics , Ribonucleases/metabolism , Ribonucleoproteins/chemistry , Ribonucleoproteins/genetics , Ribonucleoproteins/metabolism
12.
Nucleic Acids Res ; 42(8): 5332-46, 2014 Apr.
Article in English | MEDLINE | ID: mdl-24561806

ABSTRACT

The carbon storage regulator/regulator of secondary metabolism (Csr/Rsm) type of small non-coding RNAs (sRNAs) is widespread throughout bacteria and acts by sequestering the global translation repressor protein CsrA/RsmE from the ribosome binding site of a subset of mRNAs. Although we have previously described the molecular basis of a high affinity RNA target bound to RsmE, it remains unknown how other lower affinity targets are recognized by the same protein. Here, we have determined the nuclear magnetic resonance solution structures of five separate GGA binding motifs of the sRNA RsmZ of Pseudomonas fluorescens in complex with RsmE. The structures explain how the variation of sequence and structural context of the GGA binding motifs modulate the binding affinity for RsmE by five orders of magnitude (∼10 nM to ∼3 mM, Kd). Furthermore, we see that conformational adaptation of protein side-chains and RNA enable recognition of different RNA sequences by the same protein contributing to binding affinity without conferring specificity. Overall, our findings illustrate how the variability in the Csr/Rsm protein-RNA recognition allows a fine-tuning of the competition between mRNAs and sRNAs for the CsrA/RsmE protein.


Subject(s)
Bacterial Proteins/chemistry , RNA, Small Untranslated/chemistry , RNA-Binding Proteins/chemistry , 5' Untranslated Regions , Bacterial Proteins/metabolism , Models, Molecular , Nucleotide Motifs , Nucleotides/chemistry , Protein Binding , Protein Biosynthesis , Protein Conformation , Pseudomonas fluorescens/genetics , RNA, Small Untranslated/metabolism , RNA-Binding Proteins/metabolism
13.
Proc Natl Acad Sci U S A ; 110(46): 18680-5, 2013 Nov 12.
Article in English | MEDLINE | ID: mdl-24158483

ABSTRACT

The Bombyx mori pheromone-binding protein (BmorPBP) is known to adopt two different conformations. These are BmorPBP(A), where a regular helix formed by the C-terminal dodecapeptide segment, α7, occupies the ligand-binding cavity, and BmorPBP(B), where the binding site is free to accept ligands. NMR spectra of delipidated BmorPBP solutions at the physiological pH of the bulk sensillum lymph near pH 6.5 show only BmorPBP(A), and in mixtures, the two species are in slow exchange on the chemical shift frequency scale. This equilibrium has been monitored at variable pH and ligand concentrations, demonstrating that it is an intrinsic property of BmorPBP that is strongly affected by pH variation and ligand binding. This polymorphism tunes BmorPBP for optimal selective pheromone transport: Competition between α7 and lipophilic ligands for its binding cavity enables selective uptake of bombykol at the pore endings in the sensillum wall, whereas compounds with lower binding affinity can only be bound in the bulk sensillum lymph. After transport across the bulk sensillum lymph into the lower pH area near the dendritic membrane surface, bombykol is ejected near the receptor, whereas compounds with lower binding affinity are ejected before reaching the olfactory receptor, rendering them susceptible to degradation by enzymes present in the sensillum lymph.


Subject(s)
Bombyx/metabolism , Carrier Proteins/chemistry , Carrier Proteins/metabolism , Insect Proteins/chemistry , Insect Proteins/metabolism , Pheromones/metabolism , Protein Conformation , Smell/physiology , Animals , Biological Transport/physiology , Bombyx/physiology , Carrier Proteins/genetics , Fatty Alcohols/metabolism , Hydrogen-Ion Concentration , Insect Proteins/genetics , Intercellular Signaling Peptides and Proteins , Molecular Structure , Nuclear Magnetic Resonance, Biomolecular , Polymorphism, Genetic/genetics
14.
Chembiochem ; 14(4): 457-66, 2013 Mar 04.
Article in English | MEDLINE | ID: mdl-23362130

ABSTRACT

Current solution NMR techniques enable structural investigations of proteins in molecular particles with sizes up to several hundred kDa. However, the large molecular weight of proteins in such systems results in increased numbers of NMR signals, and the resulting spectral overlap typically imposes limitations. For multidomain proteins, segmental isotope labeling of individual domains facilitates the spectral interpretation by reducing the number of signals, but for large domains with small signal dispersion, signal overlap can persist. To overcome limitations arising from spectral overlap, we present a strategy that combines cell-free expression and ligation of the expressed proteins to produce multidomain proteins with selective amino acid-type labeling in individual domains. The bottleneck of intrinsically low cell-free expression yields of precursor molecules was overcome by introducing new fusion constructs that allowed milligram production of ligation-competent domains labeled in one or multiple amino acid types. Ligation-competent unlabeled partner domains were produced in vivo, and subsequent domain ligation was achieved by using an on-column strategy. This approach is illustrated with two multidomain RNA-binding proteins, that is, the two C-terminal RNA-recognition motifs of the human polypyrimidine tract-binding protein, and two highly homologous helix-turn-helix domains of the human glutamyl-prolyl-tRNA synthetase.


Subject(s)
Amino Acids/analysis , Amino Acyl-tRNA Synthetases/chemistry , Isotope Labeling/methods , Nuclear Magnetic Resonance, Biomolecular/methods , Polypyrimidine Tract-Binding Protein/chemistry , Humans , Protein Structure, Tertiary
15.
FEBS J ; 279(17): 3176-84, 2012 Sep.
Article in English | MEDLINE | ID: mdl-22776321

ABSTRACT

We describe Escherichia coli based cell-free production of milligram quantities of eukaryotic proteins containing native disulfide bonds. Using a previously described expression system, we systematically investigated the influence of redox potential variation in the reaction mixture and the impact of adding disulfide bond catalysts on soluble protein production. It is then shown that the optimized reaction conditions for native disulfide bond formation can be combined with the use of N-terminal fusion constructs with the GB1 domain for increased expression yields. The resulting cell-free system is suitable for stable-isotope labeling and does not require chemical pretreatment of the cell extract to stabilize the redox potential. For the human doppel protein, the mouse doppel protein and mouse interleukin-22 we obtained 0.3-0.7 mg of purified native protein per milliliter of reaction mixture. Formation of disulfide bonds was validated using the Ellman assay, and native folding of the three proteins was monitored by NMR and CD spectroscopy.


Subject(s)
Disulfides/chemistry , Protein Conformation , Proteins/chemistry , Animals , Catalysis , Cell-Free System , Electrophoresis, Polyacrylamide Gel , Escherichia coli/genetics , Humans , Mice , Nuclear Magnetic Resonance, Biomolecular , Oxidation-Reduction , Proteins/genetics
16.
J Biomol NMR ; 53(1): 43-51, 2012 May.
Article in English | MEDLINE | ID: mdl-22418693

ABSTRACT

Production of sufficient amounts of human proteins is a frequent bottleneck in structural biology. Here we describe an Escherichia coli-based cell-free system which yields mg-quantities of human proteins in N-terminal fusion constructs with the GB1 domain, which show significantly increased translation efficiency. A newly generated E. coli BL21 (DE3) RIPL-Star strain was used, which contains a variant RNase E with reduced activity and an excess of rare-codon tRNAs, and is devoid of lon and ompT protease activity. In the implementation of the expression system we used freshly in-house prepared cell extract. Batch-mode cell-free expression with this setup was up to twofold more economical than continuous-exchange expression, with yields of 0.2-0.9 mg of purified protein per mL of reaction mixture. Native folding of the proteins thus obtained is documented with 2D [(15)N,(1)H]-HSQC NMR.


Subject(s)
Cell-Free System , Cloning, Molecular/methods , Escherichia coli/metabolism , Recombinant Fusion Proteins/metabolism , Bacterial Proteins/chemistry , Bacterial Proteins/genetics , Bacterial Proteins/metabolism , Cell Extracts , Escherichia coli/genetics , Genetic Vectors , Humans , Isotope Labeling , Nuclear Magnetic Resonance, Biomolecular , Recombinant Fusion Proteins/chemistry , Recombinant Fusion Proteins/genetics
17.
PLoS One ; 6(7): e21799, 2011.
Article in English | MEDLINE | ID: mdl-21789181

ABSTRACT

Interleukin (IL)-22 is an effector cytokine, which acts primarily on epithelial cells in the skin, gut, liver and lung. Both pro- and anti-inflammatory properties have been reported for IL-22 depending on the tissue and disease model. In a murine model of allergic airway inflammation, we found that IL-22 is predominantly produced by innate lymphoid cells in the inflamed lungs, rather than TH cells. To determine the impact of IL-22 on airway inflammation, we used allergen-sensitized IL-22-deficient mice and found that they suffer from significantly higher airway hyperreactivity upon airway challenge. IL-22-deficiency led to increased eosinophil infiltration lymphocyte invasion and production of CCL17 (TARC), IL-5 and IL-13 in the lung. Mice treated with IL-22 before antigen challenge displayed reduced expression of CCL17 and IL-13 and significant amelioration of airway constriction and inflammation. We conclude that innate IL-22 limits airway inflammation, tissue damage and clinical decline in allergic lung disease.


Subject(s)
Immunity, Innate/immunology , Inflammation/complications , Inflammation/immunology , Interleukins/biosynthesis , Lymphocytes/immunology , Respiratory Hypersensitivity/complications , Respiratory Hypersensitivity/immunology , Allergens/immunology , Animals , Biomarkers/metabolism , Epithelial Cells/drug effects , Epithelial Cells/metabolism , Immunity, Innate/drug effects , Immunization , Immunoglobulins/blood , Inflammation/blood , Inflammation/pathology , Interleukin-13/pharmacology , Interleukins/administration & dosage , Interleukins/deficiency , Interleukins/metabolism , Intracellular Space/drug effects , Intracellular Space/metabolism , Lung/drug effects , Lung/immunology , Lung/pathology , Lymphocytes/drug effects , Mice , Phosphorylation/drug effects , Recombinant Proteins/administration & dosage , Recombinant Proteins/pharmacology , Respiratory Hypersensitivity/blood , STAT3 Transcription Factor/metabolism , T-Lymphocytes/drug effects , T-Lymphocytes/immunology , Tumor Necrosis Factor-alpha/pharmacology , Interleukin-22
18.
J Mol Biol ; 408(5): 922-31, 2011 May 20.
Article in English | MEDLINE | ID: mdl-21396939

ABSTRACT

The Bombyx mori pheromone-binding protein (BmorPBP) undergoes a pH-dependent conformational transition from a form at basic pH, which contains an open cavity suitable for ligand binding (BmorPBP(B)), to a form at pH 4.5, where this cavity is occupied by an additional helix (BmorPBP(A)). This helix α7 is formed by the C-terminal dodecapeptide 131-142, which is flexibly disordered on the protein surface in BmorPBP(B) and in its complex with the pheromone bombykol. Previous work showed that the ligand-binding cavity cannot accommodate both bombykol and helix α7. Here we further investigated mechanistic aspects of the physiologically crucial ejection of the ligand at lower pH values by solution NMR studies of the variant protein BmorPBP(1-128), where the C-terminal helix-forming tetradecapeptide is removed. The NMR structure of the truncated protein at pH 6.5 corresponds closely to BmorPBP(B). At pH 4.5, BmorPBP(1-128) maintains a B-type structure that is in a slow equilibrium, on the NMR chemical shift timescale, with a low-pH conformation for which a discrete set of (15)N-(1)H correlation peaks is NMR unobservable. The full NMR spectrum was recovered upon readjusting the pH of the protein solution to 6.5. These data reveal dual roles for the C-terminal tetradecapeptide of BmorPBP in the mechanism of reversible pheromone binding and transport, where it governs dynamic equilibria between two locally different protein conformations at acidic pH and competes with the ligand for binding to the interior cavity.


Subject(s)
Carrier Proteins/chemistry , Insect Proteins/chemistry , Amino Acid Sequence , Animals , Intercellular Signaling Peptides and Proteins , Ligands , Magnetic Resonance Spectroscopy , Molecular Sequence Data , Protein Binding , Protein Conformation , Sex Attractants/chemistry
19.
Arch Biochem Biophys ; 439(2): 234-40, 2005 Jul 15.
Article in English | MEDLINE | ID: mdl-15978540

ABSTRACT

The kinetics of bovine Cu,Zn superoxide dismutase were studied by pulse radiolysis. To ensure the absence of catalytically active free copper, commercially obtained holo-superoxide dismutase was demetallated, and the apo-superoxide dismutase concentrations were determined by isothermal titration calorimetry prior to reconstitution with defined amounts of copper and zinc. The catalytic rate constant was determined as a function of ionic strength over the range of 4-154 mM, and of the copper and zinc content. The catalytic rate constant increases with ionic strength up to (1.5 +/- 0.2) x 10(9) M(-1) s(-1) at an ionic strength of 15 mM, and then decreases. At pH 7 and 50 mM ionic strength, k = (1.2 +/- 0.2) x 10(9) M(-1) s(-1), and at a physiologically relevant ionic strength of 150 mM, it is (0.7 +/- 0.1) x 10 (9) M(-1) s(-1). The effect of ionic strength is ascribed to the inhomogeneous electric field generated by the surface charges of superoxide dismutase. The value of the catalytic rate constant at 50 mM is ca. 2-fold smaller than earlier values reported in the literature. The relationship between copper content and the catalytic rate constant shows that addition of more than a stoichiometric amount of copper cannot be masked efficiently by EDTA. The possibility exists that earlier reported values were based on experiments contaminated with trace amounts of copper.


Subject(s)
Copper/metabolism , Superoxide Dismutase/metabolism , Zinc/metabolism , Animals , Cattle , Copper/chemistry , Edetic Acid/chemistry , Kinetics , Osmolar Concentration , Pulse Radiolysis , Superoxide Dismutase/chemistry , Zinc/chemistry
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