Target Protein Of Interest Research Articles

Novel immune response to gluten in individuals with schizophrenia

A link between celiac disease and schizophrenia has been postulated for several years, based primarily on reports of elevated levels of antibody to gliadin in patients. We sought to examine the proposed connection between schizophrenia and celiac disease by characterizing the molecular specificity and mechanism of the anti-gliadin immune response in a subset of individuals with schizophrenia. Blood samples from individuals with schizophrenia and elevated anti-gliadin antibody titer were examined for celiac disease-associated biomarkers, including antibodies to transglutaminase 2 (TG2) enzyme and deamidated gliadin peptides, as well as the HLA-DQ2 and -DQ8 MHC genes. The anti-gliadin antibody response was further characterized through examination of reactivity towards chromatographically separated gluten proteins. Target proteins of interest were identified by peptide mass mapping. In contrast to celiac disease patients, an association between the anti-gliadin immune response and anti-TG2 antibody or HLA-DQ2 and -DQ8 markers was not found in individuals with schizophrenia. In addition, the majority of individuals with schizophrenia and anti-gliadin antibody did not exhibit antibody reactivity to deamidated gliadin peptides. Further characterization of the antibody specificity revealed preferential reactivity towards different gluten proteins in the schizophrenia and celiac disease groups. These findings indicate that the anti-gliadin immune response in schizophrenia has a different antigenic specificity from that in celiac disease and is independent of the action of transglutaminase enzyme and HLA-DQ2/DQ8. Meanwhile, the presence of elevated levels of antibodies to specific gluten proteins points to shared immunologic abnormalities in a subset of schizophrenia patients. Further characterization and understanding of the immune response to gluten in schizophrenia may provide novel insights into the etiopathogenesis of specific disease phenotypes.

Protein Sample Treatment with Peptide Ligand Library: Coverage and Consistency

Low-abundance protein detection in biological samples is one of the main challenges in proteomics investigations. One approach that makes the detection of rare species possible is the treatment of biological samples with solidphase combinatorial peptide ligand libraries. However, the use of combinations of ligands opens an uncertainty in that, since the diversity of the library is very large, aliquots of beads sampled from the library might not have fully comparable bead species each time. Reproducibility of experimental data with highly diverse libraries is therefore a main concern to address. This paper reports reproducibility data when aliquots of similar and different volumes of libraries are used at a certain sample to library ratio. Eluates from ligand libraries and other fractions are analyzed using various complementary methods such as two-dimensional gel electrophoresis, immunoassay and mass spectrometry. The collected data show a high level of consistency from sample to sample when processed with similar and variable bead volumes. Analytical determinations are all convergent with each other in considering the similarity of results. It is anticipated that this demonstration reinforces the possibility that differential proteomics studies, in particular for the discovery of protein targets of interest, can effectively be accomplished with combinatorial peptide libraries.

Single‐step affinity purification of recombinant proteins using a self‐excising module from Neisseria meningitidis FrpC

Purification of recombinant proteins is often a challenging process involving several chromatographic steps that must be optimized for each target protein. Here, we developed a self-excising module allowing single-step affinity chromatography purification of untagged recombinant proteins. It consists of a 250-residue-long self-processing module of the Neisseria meningitidis FrpC protein with a C-terminal affinity tag. The N terminus of the module is fused to the C terminus of a target protein of interest. Upon binding of the fusion protein to an affinity matrix from cell lysate and washing out contaminating proteins, site-specific cleavage of the Asp-Pro bond linking the target protein to the self-excising module is induced by calcium ions. This results in the release of the target protein with only a single aspartic acid residue added at the C terminus, while the self-excising affinity module remains trapped on the affinity matrix. The system was successfully tested with several target proteins, including glutathione-S-transferase, maltose-binding protein, beta-galactosidase, chloramphenicol acetyltransferase, and adenylate cyclase, and two different affinity tags, chitin-binding domain or poly-His. Moreover, it was demonstrated that it can be applied as an alternative to two currently existing systems, based on the self-splicing intein of Saccharomyces cerevisiae and sortase A of Staphylococcus aureus.

Ribosomal Synthesis of N-Methyl Peptides

N-Methyl amino acids (N-Me AAs) are a common component of nonribosomal peptides (NRPs), a class of natural products from which many clinically important therapeutics are obtained. N-Me AAs confer peptides with increased conformational rigidity, membrane permeability, and protease resistance. Hence, these analogues are highly desirable building blocks in the ribosomal synthesis of unnatural peptide libraries, from which functional, NRP-like molecules may be identified. By supplementing a reconstituted Escherichia coli translation system with specifically aminoacylated total tRNA that has been chemically methylated, we have identified three N-Me AAs (N-Me Leu, N-Me Thr, and N-Me Val) that are efficiently incorporated into peptides by the ribosome. Moreover, we have demonstrated the synthesis of peptides containing up to three N-Me AAs, a number comparable to that found in many NRP drugs. With improved incorporation efficiency and translational fidelity, it may be possible to synthesize combinatorial libraries of peptides that contain multiple N-Me AAs. Such libraries could be subjected to in vitro selection methods to identify drug-like, high-affinity ligands for protein targets of interest.

Resolving the Network of Cell Signaling Pathways using the Evolving Yeast Two-Hybrid System

In 1983, while investigators had identified a few human proteins as important regulators of specific biological outcomes, how these proteins acted in the cell was essentially unknown in almost all cases. Twenty-five years later, our knowledge of the mechanistic basis of protein action has been transformed by our increasingly detailed understanding of protein-protein interactions, which have allowed us to define cellular machines. The advent of the yeast two-hybrid (Y2H) system in 1989 marked a milestone in the field of proteomics. Exploiting the modular nature of transcription factors, the Y2H system allows facile measurement of the activation of reporter genes based on interactions between two chimeric or "hybrid" proteins of interest. After a decade of service as a leading platform for individual investigators to use in exploring the interaction properties of interesting target proteins, the Y2H system has increasingly been applied in high-throughput applications intended to map genome-scale protein-protein interactions for model organisms and humans. Although some significant technical limitations apply, Y2H has made a great contribution to our general understanding of the topology of cellular signaling networks.

Ligand discovery on massively parallel systems

Virtual screening is an approach for identifying promising leads for drugs and is used in the pharmaceutical industry. We present the parallelization of LIDAEUS (LIgand Discovery At Edinburgh UniverSity), creating a massively parallel high-throughput virtual-screening code. This program is being used to predict the binding modes involved in the docking of small ligands to proteins. Parallelization efforts have focused on achieving maximum parallel efficiency and developing a memory-efficient parallel sorting routine. Using an IBM Blue Gene/L™ supercomputer, runtimes have been reduced from 8 days on a modest seven-node cluster to 62 minutes on 1,024 processors using a standard dataset of 1.67 million small molecules and FKBP12, a protein target of interest in immunosuppressive therapies. Using more-complex datasets, the code scales upward to make use of the full processor set of 2,048. The code has been successfully used for the task of gathering data on approximately 1.67 million small molecules binding to approximately 400 high-quality crystallographically determined ligand-bound protein structures, generating data on more than 646 million protein-ligand complexes. A number of novel ligands have already been discovered and validated experimentally.

Flow cytometer with mass spectrometer detection for massively multiplexed single-cell biomarker assay

Abstract This paper describes the development and application of new metal-tagging reagents and a novel mass spectrometer (MS) detector for a flow cytometer that enables highly multiplexed measurement of many biomarkers in individual cells. A new class of tagging reagents, based on an acrylic polymer backbone that incorporates a reproducible number of lanthanide elements, has been developed. When linked to antibodies that specifically recognize target proteins of interest, determination of the tag elements is diagnostic for the presence and quantification of the antigen. The use of enriched stable isotope tags provides the opportunity for multiparametric assay. The new instrument uses inductively coupled plasma (ICP) to vaporize, atomize, and ionize individual cells that have been probed using the metal-labeled antibodies. The elemental composition, specifically of the metal tags, is recorded simultaneously using a time-of-flight (TOF)-MS that has been specifically designed for high-speed analysis during the short transient corresponding to the individual cell event. The detector provides for well-resolved atomic fingerprints of many elemental and isotopic tags, with little overlap of neighboring signals (high abundance sensitivity) and wide dynamic range both for a single antigen and between antigens.

A combined bioinformatics and chemoinformatics approach for the development of new antiparasitic drugs

A modern concept for the development of novel antiparasitic drugs is the combination of bioinformatics and chemoinformatics approaches. This covers, for example, the identification of target proteins serving as molecular points of attack for parasiticides—the idea is that, owing to some essential role, inhibition of a target protein should eradicate the parasite. To prevent toxicity problems for vertebrate host organisms, it is advantageous that these proteins show significant differences from their vertebrate counterparts. In the present work, we identified potential target proteins in parasitic nematodes ( Ascaris suum, Brugia malayi, and Haemonchus contortus) and arthropods ( Boophilus microplus and Rhipicephalus appendiculatus) using bioinformatic sequence comparison methods on expressed sequence tags. Interesting target proteins (e.g., S-adenosyl- l-methionine synthetase) were characterized in detail by subjecting them to in-depth bioinformatic analysis. S-Adenosyl- l-methionine synthetase was also used to elucidate chemoinformatics approaches like homology modeling and docking, which represent appropriate methods for generating valuable data for the development of new drug candidates.

Utility of cleavable isotope-coded affinity-tagged reagents for quantification of low-copy proteins induced by methylprednisolone using liquid chromatography/tandem mass spectrometry.

Gene expression changes underlie important biological and pharmacological responses. Although mRNA expression profiling is routine, quantification of low-abundance proteins, which typically represent key effectors of responses, remains challenging. A novel strategy was developed for sensitive and accurate quantification of low-abundance proteins in highly complex biological matrixes. First, the cysteine specificity of cleavable isotope-coded affinity tags (cICAT) was employed to reduce the complexity of the digested proteome of tissue homogenates and to improve the quantification of low-abundance proteins. Second, cICAT-treated tissue samples were analyzed on a capillary LC coupled to an ion trap MS to screen for the subset of cICAT-peptides, derived from target proteins of interest, that was successfully labeled and retrieved. Third, putatively identified peptides derived from target proteins were synthesized, cICAT-labeled, and used both to optimize multiple reactions monitoring (MRM) analysis and to confirm chromatographic retention time and fragmentation pattern. Finally, batch quantification of target peptides was performed using MRM on a LC/triple-quad MS/MS using (12)C- (control) and (13)C (experimental)-cICAT-labeled tissue mixtures. The utility of this method was demonstrated by elucidating the time-course of tyrosine aminotransferase induction in the liver of rats following treatment with the corticosteroid methylprednisolone (MPL). This approach significantly improved quantitative sensitivity, and the linear range was 10-fold greater than published previously. An additional advantage is that archived samples may be reinterrogated to investigate the regulation of additional targets that become of interest. Stored samples were sucessfully reinterrogated to monitor the induction of ornithine decarboxylase, which is also an MPL-induced protein. To our knowledge, this is the first report of an ICAT-based method that is capable of quantifying low-abundance proteins in highly complex samples, such as tissue homogenates. The approach enables simultaneous quantification of multiple effector proteins induced by biological or pharmacological stimuli, and the processed samples can be interrogated repeatedly as additional targets of interest arise.

Protein Ligand Design: From Phage Display to Synthetic Protein Epitope Mimetics in Human Antibody Fc-Binding Peptidomimetics

Phage display is a powerful method for selecting peptides with novel binding functions. Synthetic peptidomimetic chemistry is a powerful tool for creating structural diversity in ligands as a means to establish structure-activity relationships. Here we illustrate a method of bridging these two methodologies, by starting with a disulfide bridged phage display peptide which binds a human antibody Fc fragment (Delano et al. Science 2000, 287, 1279) and creating a backbone cyclic beta-hairpin peptidomimetic with 80-fold higher affinity for the Fc domain. The peptidomimetic is shown to adopt a well-defined beta-hairpin conformation in aqueous solution, with a bulge in one beta-strand, as seen in the crystal structure of the phage peptide bound to the Fc domain. The higher binding affinity of the peptidomimetic presumably reflects the effect of constraining the free ligand into the conformation required for binding, thus highlighting in this example the influence that ligand flexibility has on the binding energy. Since phage display peptides against a wide variety of different proteins are now accessible, this approach to synthetic ligand design might be applied to many other medicinally and biotechnologically interesting target proteins.

Identification of preferred protein interactions by phage‐display of the human Src homology‐3 proteome

We have determined the human genome to contain 296 different Src homology-3 (SH3) domains and cloned them into a phage-display vector. This provided a powerful and unbiased system for simultaneous assaying of the complete human SH3 proteome for the strongest binding to target proteins of interest, without the limitations posed by short linear peptide ligands or confounding variables of more indirect methods for protein interaction screening. Studies involving three ligand proteins, human immunodeficiency virus-1 Nef, p21-activated kinase (PAK)2 and ADAM15, showed previously reported as well as novel SH3 partners with nanomolar affinities specific for them. This argues that SH3 domains may have a more dominant role in directing cellular protein interactions than has been assumed. Besides showing potentially important new SH3-directed interactions, these studies also led to the discovery of novel signalling proteins, such as the PAK2-binding adaptor protein POSH2 and the ADAM15-binding sorting nexin family member SNX30.

Multiple Reaction Monitoring to Identify Sites of Protein Phosphorylation with High Sensitivity

Phosphorylation governs the activity of many proteins. Insight into molecular mechanisms in biology would be immensely improved by robust, sensitive methods for identifying precisely sites of phosphate addition. An approach to selective mapping of protein phosphorylation sites on a specific target protein of interest using LC-MS is described here. In this approach multiple reaction monitoring is used as an extremely sensitive MS survey scan for potential phosphopeptides from a known protein. This is automatically followed by peptide sequencing and subsequent location of the phosphorylation site; both of these steps occur in a single LC-MS run, providing greater efficiency of sample use. The method is capable of detecting and sequencing phosphopeptides at low femtomole levels with high selectivity. As proof of the value of this approach in an experimental setting, a key Schizosaccharomyces pombe cell cycle regulatory protein, Cyclin B, was purified, and associated proteins were identified. Phosphorylation sites on these proteins were located. The technique, which we have called multiple reaction monitoring-initiated detection and sequencing (MIDAS), is shown to be a highly sensitive approach to the determination of protein phosphorylation.

Coming of age: carbohydrates and immunity

Adaptive immune responses have long been considered the "territory" of antigenic proteins, whereas carbohydrates are characterized as T-cell-independent antigens that are not typically recognized by the complete adaptive machinery. The current modus operandi when searching for dominant epitopes is the use of synthetic peptides designed from the primary structure of interesting target proteins; however, there is growing evidence that sugars can also play a critical role in immune recognition. Findings reported in this issue of the European Journal of Immunology begin to shed light on the differences in protein glycosylation that can occur in association with disorders like rheumatoid arthritis and the effect these changes have on collagen recognition by the immune system. Other recent studies have shown that immunodominant glycopeptide "remnant" epitopes as well as glycosylation changes on self-proteins can generate autoimmunity. Finally, some types of carbohydrates are now known to be processed and presented to T cells by class II MHC. Taken together, these advances illustrate a clear importance for carbohydrate recognition in foreign and self antigens by the adaptive immune system. With the common presence of carbohydrate molecules on eukaryotic, prokaryotic, and viral surfaces, the impact of carbohydrates on adaptive immunity is now indisputable.

Refinement of the conformation of UDP-galactose bound to galactosyltransferase using the STD NMR intensity-restrained CORCEMA optimization.

The STD NMR technique has originally been described as a tool for screening large compound libraries to identify the lead compounds that are specific to target proteins of interest. The application of this technique in the qualitative epitope mapping of ligands weakly binding to proteins, virus capsid shells, and nucleic acids has also been described. Here we describe the application of the STD NMR intensity-restrained CORCEMA optimization (SICO) procedure for refining the bound conformation of UDP-galactose in galactosyltransferase complex using STD NMR intensities recorded at 500 MHz as the experimental constraints. A comparison of the SICO structure for the bound UDP-galactose in solution with that in the crystal structure for this complex shows some differences in ligand torsion angles and V253 side-chain orientation in the protein. This work describes the first application of an STD NMR intensity-restrained CORCEMA optimization procedure for refining the torsion angles of a bound ligand structure. This method is likely to be useful in structure-based drug design programs since most initial lead compounds generally exhibit weak affinity (millimolar to micromolar) to target proteins of pharmaceutical interest, and the bound conformation of these lead compounds in the protein binding pocket can be determined by the CORCEMA-ST refinement.

Nanoparticle-based bio-bar codes for the ultrasensitive detection of proteins.

An ultrasensitive method for detecting protein analytes has been developed. The system relies on magnetic microparticle probes with antibodies that specifically bind a target of interest [prostate-specific antigen (PSA) in this case] and nanoparticle probes that are encoded with DNA that is unique to the protein target of interest and antibodies that can sandwich the target captured by the microparticle probes. Magnetic separation of the complexed probes and target followed by dehybridization of the oligonucleotides on the nanoparticle probe surface allows the determination of the presence of the target protein by identifying the oligonucleotide sequence released from the nanoparticle probe. Because the nanoparticle probe carries with it a large number of oligonucleotides per protein binding event, there is substantial amplification and PSA can be detected at 30 attomolar concentration. Alternatively, a polymerase chain reaction on the oligonucleotide bar codes can boost the sensitivity to 3 attomolar. Comparable clinically accepted conventional assays for detecting the same target have sensitivity limits of approximately 3 picomdar, six orders of magnitude less sensitive than what is observed with this method.

Mapping Protein‐Protein Interactions with Phage‐Displayed Combinatorial Peptide Libraries

This unit describes the process and analysis of affinity selecting bacteriophage M13 from libraries displaying combinatorial peptides fused to either a minor or major capsid protein. Direct affinity selection uses target protein bound to a microtiter plate followed by purification of selected phage by ELISA. Alternatively, there is a bead-based affinity selection method. These methods allow one to readily isolate peptide ligands that bind to a protein target of interest and use the consensus sequence to search proteomic databases for putative interacting proteins.

Application of Random Peptide Phage Display to the Study of Nuclear Hormone Receptors

This chapter focuses on the application of random peptide phage display to the study of nuclear hormone receptors. The chapter is organized into four sections: (1) the construction of a peptide phage display library, (2) a step-by-step protocol for biopanning (the affinity selection process), (3) the discussion of a mammalian cell-based screening technology to validate and complement the in vitro phage display approach, and (4) the use of phage display selected peptides as antagonists of nuclear receptor function. The selection of peptides that bind to the target proteins of interest is achieved by incubating phage libraries containing small random peptide inserts with the target proteins that are immobilized on a solid support. Phage display has been widely used as a method to map protein–protein interactions, identify peptide ligands for cell surface receptors, and map antibody–antigen epitopes. By using phage display, random peptide libraries can be generated and specific target-interacting peptides can be identified. The random peptide libraries are created by inserting short random oligonucleotides within the coding sequence of the M13 bacteriophage capsid protein, with the subsequent display of random peptides on the outer surface of the phage. In addition to using phage display to study receptor conformation and SERM pharmacology, this powerful technique has been used to develop highly specific peptide antagonists that block receptor function by interfering with required protein–protein interactions. A very good alternative to immobilizing steroid–nuclear hormone receptors and other DNA-binding transcription factors is by using biotinylated oligonucleotides (corresponding to their cognate response element) pre-captured on streptavidin-coated plates.

6] Photophysics of green and red fluorescent proteins: Implications for quantitative microscopy

This chapter describes the implications for quantitative microscopy. The green fluorescent protein (GFP) from the jellyfish Aequorea victoria and its mutants constitute a class of fluorophores that has revolutionized the evaluation of molecular interactions and visualization of biological systems. When fused to proteins of interest and expressed in vivo, fluorescent proteins (FPs) act as versatile indicators of structure and function within cells and can be imaged with the full repertoire of fluorescence microscopy techniques. FPs and their constructs are finding increasing use in fluorescence lifetime imaging microscopy (FLIM) and fluorescence resonance energy transfer (FRET) modes of microspectroscopy for the elucidation of protein-protein interactions, signaling, and trafficking in cellular systems. The simple and the most common, application of FPs is as a passive marker fused to a target protein of interest for visualizing its spatiotemporal distribution. Mutations in and around the chromophore have systematic effects on the spectra. Green and red fluorescent proteins exhibit photophysical properties that are generally more complex than those of traditional fluorophores, for example, fluorescein and rhodamine.

Mapping protein‐protein interactions with combinatorial peptides

Now that a number of genomes have been sequenced, attention has turned to understanding the complement of proteins encoded by the genome, which has been termed the ‘proteome’. One important aspect of analysis of the proteome is the identification of which proteins interact with each other; this information is invaluable in surmising the function of each protein and inferring what cellular pathway the protein may be a component of. While a variety of methodologies is widely used to map protein-protein interactions, such as affinity purification followed by mass spectrometry and yeast two-hybrid screening (both of which were covered at this meeting), an alternative approach is the use of phage-displayed combinatorial peptides. In phage-display, short oligonucleotides are inserted within a gene encoding a capsid (coat) protein of a bacteriophage, so that each viral particle displays a different peptide sequence. While it has been possible to clone and express short peptides attached to each of the five different capsid proteins of bacteriophage M13, the protein products of gene III and VIII are popular cloning sites for expression and display. Several excellent reviews of phage-display can be found elsewhere [6,22,5]. Over the past ten years, a considerable number of phage-displayed combinatorial peptide libraries have been generated [21]. With a library of recombinant phage particles in hand, it is possible to screen them by affinity selection with a variety of protein targets. Three rounds of selection (binding, elution, propagation) are typically sufficient to screen billions of different phage-displayed combinatorial peptides for those that bind to target proteins of interest. While there have been many recent publications of successful selection experiments, some of the more notable examples are the selection of peptide ligands which bind to receptors for erythropoietin [30], N-methyl D-aspartate (NMDA) [16], thrombopoietin [7], fibroblast growth factor [3], and estrogen [17]. When peptides are chemically synthesized corresponding to what is displayed by the selected phage, they generally bind with dissociation constant (Kd) values of 5 micromolar to 10 nanomolar to their cognate receptor, and typically have agonist or antagonist activities. Many different types of targets will yield phage after affinity selection, such as enzymes [12], growth factors [10,4], nucleic acids [2], and cells [20,13]. Comparative and Functional Genomics Comp Funct Genom 2001; 2: 304–306. DOI: 10.1002 / cfg.100

The Tandem Affinity Purification (TAP) Method: A General Procedure of Protein Complex Purification

Identification of components present in biological complexes requires their purification to near homogeneity. Methods of purification vary from protein to protein, making it impossible to design a general purification strategy valid for all cases. We have developed the tandem affinity purification (TAP) method as a tool that allows rapid purification under native conditions of complexes, even when expressed at their natural level. Prior knowledge of complex composition or function is not required. The TAP method requires fusion of the TAP tag, either N- or C-terminally, to the target protein of interest. Starting from a relatively small number of cells, active macromolecular complexes can be isolated and used for multiple applications. Variations of the method to specifically purify complexes containing two given components or to subtract undesired complexes can easily be implemented. The TAP method was initially developed in yeast but can be successfully adapted to various organisms. Its simplicity, high yield, and wide applicability make the TAP method a very useful procedure for protein purification and proteome exploration.