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1.
Nat Commun ; 10(1): 365, 2019 01 21.
Article in English | MEDLINE | ID: mdl-30664649

ABSTRACT

Wnt-induced ß-catenin-mediated transcription is a driving force for stem cell self-renewal during adult tissue homeostasis. Enhanced Wnt receptor expression due to mutational inactivation of the ubiquitin ligases RNF43/ZNRF3 recently emerged as a leading cause for cancer development. Consequently, targeting canonical Wnt receptors such as LRP5/6 holds great promise for treatment of such cancer subsets. Here, we employ CIS display technology to identify single-domain antibody fragments (VHH) that bind the LRP6 P3E3P4E4 region with nanomolar affinity and strongly inhibit Wnt3/3a-induced ß-catenin-mediated transcription in cells, while leaving Wnt1 responses unaffected. Structural analysis reveal that individual VHHs variably employ divergent antigen-binding regions to bind a similar surface in the third ß-propeller of LRP5/6, sterically interfering with Wnt3/3a binding. Importantly, anti-LRP5/6 VHHs block the growth of Wnt-hypersensitive Rnf43/Znrf3-mutant intestinal organoids through stem cell exhaustion and collective terminal differentiation. Thus, VHH-mediated targeting of LRP5/6 provides a promising differentiation-inducing strategy for treatment of Wnt-hypersensitive tumors.


Subject(s)
Low Density Lipoprotein Receptor-Related Protein-5/chemistry , Low Density Lipoprotein Receptor-Related Protein-6/chemistry , Organoids/drug effects , Single-Domain Antibodies/chemistry , Stem Cells/drug effects , Wnt3A Protein/genetics , Animals , Binding Sites , Cell Differentiation , Cell Line, Tumor , Cell Proliferation , Crystallography, X-Ray , Fibroblasts/cytology , Fibroblasts/drug effects , Fibroblasts/metabolism , Gene Expression Regulation , HEK293 Cells , Humans , Intestine, Small/cytology , Intestine, Small/drug effects , Intestine, Small/metabolism , Low Density Lipoprotein Receptor-Related Protein-5/antagonists & inhibitors , Low Density Lipoprotein Receptor-Related Protein-5/genetics , Low Density Lipoprotein Receptor-Related Protein-5/metabolism , Low Density Lipoprotein Receptor-Related Protein-6/antagonists & inhibitors , Low Density Lipoprotein Receptor-Related Protein-6/genetics , Low Density Lipoprotein Receptor-Related Protein-6/metabolism , Mice , Models, Molecular , Organoids/cytology , Organoids/metabolism , Protein Binding , Protein Conformation, beta-Strand , Protein Interaction Domains and Motifs , Single-Domain Antibodies/genetics , Single-Domain Antibodies/metabolism , Stem Cells/cytology , Stem Cells/metabolism , Transcription, Genetic , Ubiquitin-Protein Ligases/genetics , Ubiquitin-Protein Ligases/metabolism , Wnt3A Protein/metabolism , beta Catenin/genetics , beta Catenin/metabolism
2.
Sci Rep ; 8(1): 16292, 2018 11 02.
Article in English | MEDLINE | ID: mdl-30389954

ABSTRACT

Bacterial surfaces are decorated with carbohydrate structures that may serve as ligands for host receptors. Based on their ability to recognize specific sugar epitopes, plant lectins are extensively used for bacteria typing. We previously observed that the galactose-specific agglutinins from Ricinus communis (RCA) and Viscum album (VAA) exhibited differential binding to nontypeable Haemophilus influenzae (NTHi) clinical isolates, their binding being distinctly affected by truncation of the lipooligosaccharide (LOS). Here, we examined their binding to the structurally similar LOS molecules isolated from strains NTHi375 and RdKW20, using microarray binding assays, saturation transfer difference NMR, and molecular dynamics simulations. RCA bound the LOSRdKW20 glycoform displaying terminal Galß(1,4)Glcß, whereas VAA recognized the Galα(1,4)Galß(1,4)Glcß epitope in LOSNTHi375 but not in LOSRdKW20, unveiling a different presentation. Binding assays to whole bacterial cells were consistent with LOSNTHi375 serving as ligand for VAA, and also suggested recognition of the glycoprotein HMW1. Regarding RCA, comparable binding to NTHi375 and RdKW20 cells was observed. Interestingly, an increase in LOSNTHi375 abundance or expression of HMW1 in RdKW20 impaired RCA binding. Overall, the results revealed that, besides the LOS, other carbohydrate structures on the bacterial surface serve as lectin ligands, and highlighted the impact of the specific display of cell surface components on lectin binding.


Subject(s)
Antigens, Bacterial/metabolism , Bacterial Typing Techniques/methods , Haemophilus influenzae/immunology , Lipopolysaccharides/metabolism , Plant Lectins/metabolism , Antigens, Bacterial/immunology , Biological Assay/methods , Galactose/metabolism , Haemophilus influenzae/classification , Haemophilus influenzae/metabolism , Lipopolysaccharides/immunology , Microarray Analysis/methods , Molecular Dynamics Simulation , Nuclear Magnetic Resonance, Biomolecular/methods , Plant Lectins/immunology
3.
Methods Mol Biol ; 1785: 53-63, 2018.
Article in English | MEDLINE | ID: mdl-29714011

ABSTRACT

We present a method to study the interaction between biomolecules and receptors present on the cell surface. This enables studies of molecular interactions in a natural biological context. As the analyte interacts with the receptors still intact on the cell surface, the experimental data provides complete dynamics and complexity of the interaction, thereby generating highly informative data. Attana's cell-based biosensor platform can be used to obtain this information from a diverse range of interactions as described in these protocols, which detail how to grow or capture cells on a surface, how to stabilize and visualize the cells on the surface, and how to set up assays to measure detailed interaction kinetics directly on the cell surface.


Subject(s)
Biosensing Techniques/methods , Cell Membrane/chemistry , Proteins/isolation & purification , Quartz Crystal Microbalance Techniques/methods , Cell Membrane/genetics , Humans , Kinetics , Protein Binding , Proteins/chemistry
4.
Methods Enzymol ; 598: 37-70, 2018.
Article in English | MEDLINE | ID: mdl-29306443

ABSTRACT

Bacterial surfaces are decorated with a diversity of carbohydrate structures that play important roles in the bacteria-host relationships. They may offer protection against host defense mechanisms, elicit strong antigenic responses, or serve as ligands for host receptors, including lectins of the innate immune system. Binding by these lectins may trigger defense responses or, alternatively, promote attachment, thereby enhancing infection. The outcome will depend on the particular bacterial surface landscape, which may substantially differ among species and strains. In this chapter, we describe two novel methods for exploring interactions directly on the bacterial surface, based on the generation of bacterial microarrays and quartz crystal microbalance (QCM) sensor chips. Bacterial microarrays enable profiling of accessible carbohydrate structures and screening of their recognition by host receptors, also providing information on binding avidity, while the QCM approach allows determination of binding affinity and kinetics. In both cases, the chief element is the use of entire bacterial cells, so that recognition of the bacterial glycan epitopes is explored in their natural environment.


Subject(s)
Lectins/immunology , Microarray Analysis/methods , Polysaccharides, Bacterial/immunology , Quartz Crystal Microbalance Techniques/methods , Receptors, Immunologic/immunology , Host Microbial Interactions/immunology , Kinetics , Klebsiella pneumoniae/chemistry , Klebsiella pneumoniae/immunology , Lectins/chemistry , Ligands , Microarray Analysis/instrumentation , Polysaccharides, Bacterial/chemistry , Quartz Crystal Microbalance Techniques/instrumentation , Receptors, Immunologic/chemistry
5.
Anal Chem ; 88(11): 5950-7, 2016 06 07.
Article in English | MEDLINE | ID: mdl-27176788

ABSTRACT

Recognition of bacterial surface epitopes by host receptors plays an important role in the infectious process and is intimately associated with bacterial virulence. Delineation of bacteria-host interactions commonly relies on the detection of binding events between purified bacteria- and host-target molecules. In this work, we describe a combined microarray and quartz crystal microbalance (QCM) approach for the analysis of carbohydrate-mediated interactions directly on the bacterial surface, thus preserving the native environment of the bacterial targets. Nontypeable Haemophilus influenzae (NTHi) was selected as a model pathogenic species not displaying a polysaccharide capsule or O-antigen-containing lipopolysaccharide, a trait commonly found in several important respiratory pathogens. Here, we demonstrate the usefulness of NTHi microarrays for exploring the presence of carbohydrate structures on the bacterial surface. Furthermore, the microarray approach is shown to be efficient for detecting strain-selective binding of three innate immune lectins, namely, surfactant protein D, human galectin-8, and Siglec-14, to different NTHi clinical isolates. In parallel, QCM bacteria-chips were developed for the analysis of lectin-binding kinetics and affinity. This novel QCM approach involves capture of NTHi on lectin-derivatized chips followed by formaldehyde fixation, rendering the bacteria an integrated part of the sensor chip, and subsequent binding assays with label-free lectins. The binding parameters obtained for selected NTHi-lectin pairs provide further insights into the interactions occurring at the bacterial surface.


Subject(s)
Haemophilus influenzae/chemistry , Lectins/analysis , Microarray Analysis , Polysaccharides/chemistry , Quartz Crystal Microbalance Techniques
6.
Methods Mol Biol ; 1258: 125-43, 2015.
Article in English | MEDLINE | ID: mdl-25447862

ABSTRACT

Precipitation, aggregation, and inclusion body (IB) formation are frequently observed problems upon overexpression of recombinant proteins. The open accessibility of cell-free reactions allows addressing such critical steps by the addition of protein stabilizers such as chemical chaperones or detergents directly into the expression reactions. This approach could therefore reduce or even prevent initial protein precipitation already in the translation environment. The strategy might be considered to generally improve protein sample quality and to rescue proteins that are difficult to refold from IBs or from aggregated precipitates. We describe a protocol for the co-translational stabilization of difficult proteins by their expression in the presence of supplements such as alcohols, poly-ions, or detergents. We compile potentially useful compounds together with their recommended stock and working concentrations. Examples of screening experiments in order to systematically identify compounds or compound mixtures that stabilize particular proteins of interest are given. The method can primarily be considered for the production of unstable soluble proteins or of membrane proteins containing larger soluble domains.


Subject(s)
Cell-Free System/metabolism , Protein Biosynthesis/physiology , Recombinant Proteins/metabolism , Animals , Humans , Membrane Proteins/metabolism , Protein Folding , Protein Stability
7.
Methods Mol Biol ; 1261: 171-95, 2015.
Article in English | MEDLINE | ID: mdl-25502200

ABSTRACT

Cell-free expression has emerged as a new standard for the production of membrane proteins. The reduction of expression complexity in cell-free systems eliminates central bottlenecks and allows the reliable and efficient synthesis of many different types of membrane proteins. Furthermore, the open accessibility of cell-free reactions enables the co-translational solubilization of cell-free expressed membrane proteins in a large variety of supplied additives. Hydrophobic environments can therefore be adjusted according to the requirements of individual membrane protein targets. We present different approaches for the preparative scale cell-free production of G-protein-coupled receptors using the extracts of Escherichia coli cells. We exemplify expression conditions implementing detergents, nanodiscs, or liposomes. The generated protein samples could be directly used for further functional characterization.


Subject(s)
Cell-Free System/metabolism , Receptors, G-Protein-Coupled/biosynthesis , Receptors, G-Protein-Coupled/isolation & purification , Cell Extracts/chemistry , Detergents/chemistry , Escherichia coli/metabolism , Liposomes/chemistry , Nanostructures/chemistry , Protein Binding , Protein Folding , Solubility
8.
Methods Mol Biol ; 1118: 109-30, 2014.
Article in English | MEDLINE | ID: mdl-24395412

ABSTRACT

This chapter addresses two major bottlenecks in cell-free membrane protein production. Firstly, we describe the optimization of expression templates for obtaining membrane proteins in preparative scales. We present details for a newly established tag variation screen providing high success rates in improving expression efficiencies while having only minimal impacts on the target protein structure. Secondly, we present protocols for the efficient co-translational insertion of membrane proteins into defined lipid bilayers. We describe the production of nanodiscs and their implementation into cell-free expression reactions for the co-translational reconstitution of membrane proteins. In addition we give guidelines for the loading of nanodiscs with different lipids in order to systematically analyze effects of lipids on the translocation, functional folding, and stability of cell-free expressed membrane proteins.


Subject(s)
Membrane Proteins/biosynthesis , Nanotechnology/methods , Protein Biosynthesis , Base Sequence , Cell-Free System , DNA-Directed RNA Polymerases/metabolism , Escherichia coli/cytology , Escherichia coli/genetics , Fermentation , Lipid Bilayers/chemistry , Lipid Bilayers/metabolism , Membrane Proteins/chemistry , Membrane Proteins/genetics , Membrane Proteins/metabolism , Models, Molecular , Nanotechnology/instrumentation , Plasmids/genetics , Protein Conformation , Solubility
9.
Biochim Biophys Acta ; 1828(9): 2182-92, 2013 Sep.
Article in English | MEDLINE | ID: mdl-23747296

ABSTRACT

The human endothelin receptors are members of the rhodopsin class A of G-protein coupled receptors and key modulators of blood pressure regulation. Their functional in vitro characterization has widely been limited by the availability of high quality samples. We have optimized cell-free expression protocols for the human endothelin A and endothelin B receptors by implementing co-translational association approaches of the synthesized proteins with supplied liposomes or nanodiscs. Efficiency of membrane association and ligand binding properties of the receptors have systematically been studied in correlation to different membrane environments and lipid types. Ligand binding was analyzed by a number of complementary assays including radioassays, surface plasmon resonance and fluorescence measurements. High affinity binding of the peptide ligand ET-1 to both endothelin receptors could be obtained with several conditions and the highest Bmax values were measured in association with nanodiscs. We could further obtain the characteristic differential binding pattern of the two endothelin receptors with a panel of selected agonists and antagonists. Two intrinsic properties of the functionally folded endothelin B receptor, the proteolytic processing based on conformational recognition as well as the formation of SDS-resistant complexes with the peptide ligand ET-1, were observed with samples obtained from several cell-free expression conditions. High affinity and specific binding of ligands could furthermore be obtained with non-purified receptor samples in crude cell-free reaction mixtures, thus providing new perspectives for fast in vitro screening applications.


Subject(s)
Endothelin-1/chemistry , Liposomes/chemistry , Receptor, Endothelin A/chemistry , Receptor, Endothelin B/chemistry , Cell-Free System/metabolism , Detergents/chemistry , Endothelin-1/metabolism , Gene Expression , Humans , Kinetics , Nanostructures/chemistry , Protein Binding , Protein Folding , Receptor, Endothelin A/biosynthesis , Receptor, Endothelin A/genetics , Receptor, Endothelin B/biosynthesis , Receptor, Endothelin B/genetics
10.
Mol Membr Biol ; 30(1): 75-89, 2013 Feb.
Article in English | MEDLINE | ID: mdl-22716775

ABSTRACT

Routine strategies for the cell-free production of membrane proteins in the presence of detergent micelles and for their efficient co-translational solubilization have been developed. Alternatively, the expression in the presence of rationally designed lipid bilayers becomes interesting in particular for biochemical studies. The synthesized membrane proteins would be directed into a more native-like environment and cell-free expression of transporters, channels or other membrane proteins in the presence of supplied artificial membranes could allow their subsequent functional analysis without any exposure to detergents. In addition, lipid-dependent effects on activity and stability of membrane proteins could systematically be studied. However, in contrast to the generally efficient detergent solubilization, the successful stabilization of membrane proteins with artificial membranes appears to be more difficult. A number of strategies have therefore been explored in order to optimize the co-translational association of membrane proteins with different forms of supplied lipid bilayers including liposomes, bicelles, microsomes or nanodiscs. In this review, we have compiled the current state-of-the-art of this technology and we summarize parameters which have been indicated as important for the co-translational association of cell-free synthesized membrane proteins with supplied membranes.


Subject(s)
Lipid Bilayers/metabolism , Membrane Proteins/biosynthesis , Protein Biosynthesis , Detergents/chemistry , Lipid Bilayers/chemistry , Liposomes/chemistry , Liposomes/metabolism , Membrane Proteins/chemistry , Membrane Proteins/genetics , Nanotechnology , Protein Transport
11.
Biochim Biophys Acta ; 1818(12): 3098-106, 2012 Dec.
Article in English | MEDLINE | ID: mdl-22960287

ABSTRACT

Nanodiscs (NDs) enable the analysis of membrane proteins (MP) in natural lipid bilayer environments. In combination with cell-free (CF) expression, they could be used for the co-translational insertion of MPs into defined membranes. This new approach allows the characterization of MPs without detergent contact and it could help to identify effects of particular lipids on catalytic activities. Association of MPs with different ND types, quality of the resulting MP/ND complexes as well as optimization parameters are still poorly analyzed. This study describes procedures to systematically improve CF expression protocols for the production of high quality MP/ND complexes. In order to reveal target dependent variations, the co-translational ND complex formation with the bacterial proton pump proteorhodopsin (PR), with the small multidrug resistance transporters SugE and EmrE, as well as with the Escherichia coli MraY translocase was studied. Parameters which modulate the efficiency of MP/ND complex formation have been identified and in particular effects of different lipid compositions of the ND membranes have been analyzed. Recorded force distance pattern as well as characteristic photocycle dynamics indicated the integration of functionally folded PR into NDs. Efficient complex formation of the E. coli MraY translocase was dependent on the ND size and on the lipid composition of the ND membranes. Active MraY protein could only be obtained with ND containing anionic lipids, thus providing new details for the in vitro analysis of this pharmaceutically important protein.


Subject(s)
Bacterial Proteins/chemistry , Escherichia coli Proteins/chemistry , Escherichia coli/metabolism , Rhodopsin/chemistry , Transferases/chemistry , Antiporters/chemistry , Antiporters/metabolism , Bacterial Proteins/metabolism , Escherichia coli Proteins/metabolism , Lipid Bilayers/chemistry , Lipid Bilayers/metabolism , Membrane Proteins/chemistry , Membrane Proteins/metabolism , Molecular Chaperones/chemistry , Molecular Chaperones/metabolism , Rhodopsin/metabolism , Rhodopsins, Microbial , Transferases/metabolism , Transferases (Other Substituted Phosphate Groups)
12.
Methods Mol Biol ; 800: 201-25, 2012.
Article in English | MEDLINE | ID: mdl-21964791

ABSTRACT

We describe a system for the cell-free expression of proteins based on extracts from Escherichia coli. Two reaction configurations, batch and continuous exchange, are discussed and analytical scale as well as preparative scale setups are documented. Guidelines for the systematic development and optimization of cell-free expression protocols are given in detail. We further provide specific protocols and parameters for the cell-free production of membrane proteins. High-throughput screening applications of CF expression systems are exemplified as new tools for genomics and proteomics studies.


Subject(s)
Escherichia coli/genetics , Protein Engineering/methods , Recombinant Proteins/biosynthesis , Bacteriophage T7/enzymology , Cell Fractionation , DNA/genetics , DNA/metabolism , DNA-Directed RNA Polymerases/metabolism , Detergents/chemistry , Escherichia coli/cytology , Magnesium/chemistry , Membrane Proteins/biosynthesis , Membrane Proteins/genetics , Membrane Proteins/isolation & purification , Recombinant Proteins/genetics , Recombinant Proteins/isolation & purification
13.
N Biotechnol ; 28(3): 262-71, 2011 Apr 30.
Article in English | MEDLINE | ID: mdl-20637904

ABSTRACT

Cell-free expression has emerged as a powerful technique to overcome major restrictions of classical in vivo membrane protein production, with sample yields of mgms of protein per ml reaction volume possible in less than a day. The open nature and high versatility of cell-free expression allows a variety of completely new ways to rationally design and optimise expression environments as well as to modulate folding kinetics for membrane proteins independent of their origin, size, topology and function. This article summarises the array of currently available options to modify and develop cell-free expression protocols adapted to the specific requirements of individual membrane proteins. We give further an overview of the recent advances of cell-free production of membrane proteins for structural and functional analysis.


Subject(s)
Cell-Free System/metabolism , Membrane Proteins/chemistry , Membrane Proteins/metabolism , Protein Biosynthesis , Humans , Membrane Proteins/genetics , Models, Molecular
14.
J Struct Biol ; 172(1): 94-106, 2010 Oct.
Article in English | MEDLINE | ID: mdl-20460156

ABSTRACT

G-protein coupled receptors still represent one of the most challenging targets in membrane protein research. Here we present a strategic approach for the cell-free synthesis of these complex membrane proteins exemplified by the preparative scale production of the human endothelin A receptor. The versatility of the cell-free expression system was used to modulate sample quality by alteration of detergents hence presenting different solubilization environments to the synthesized protein at different stages of the production process. Sample properties after co-translational and post-translational solubilization have been analysed by evaluation of homogeneity, protein stability and receptor ligand binding competence. This is a first quality evaluation of a membrane protein obtained in two different cell-free expression modes and we demonstrate that both can be used for the production of ligand-binding competent endothelin A receptor in quantities sufficient for structural approaches. The presented strategy of cell-free expression protocol development could serve as basic guideline for the production of related receptors in similar systems.


Subject(s)
Cell-Free System/metabolism , Receptor, Endothelin A/biosynthesis , Receptors, G-Protein-Coupled/biosynthesis , Binding, Competitive , Blotting, Western , Chromatography, Affinity , Circular Dichroism , Detergents/chemistry , Fluorescence Polarization , Humans , Ligands , Protein Stability , Proteomics/methods , Radioligand Assay , Receptor, Endothelin A/chemistry , Receptor, Endothelin A/metabolism , Receptors, G-Protein-Coupled/chemistry , Receptors, G-Protein-Coupled/metabolism , Reproducibility of Results , Solubility
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