Citraconate inhibits ACOD1 (IRG1) catalysis, reduces interferon responses and oxidative stress, and modulates inflammation and cell metabolism.

Although the immunomodulatory and cytoprotective properties of itaconate have been studied extensively, it is not known whether its naturally occurring isomers mesaconate and citraconate have similar properties. Here, we show that itaconate is partially converted to mesaconate intracellularly and that mesaconate accumulation in macrophage activation depends on prior itaconate synthesis. When added to human cells in supraphysiological concentrations, all three isomers reduce lactate levels, whereas itaconate is the strongest succinate dehydrogenase (SDH) inhibitor. In cells infected with influenza A virus (IAV), all three isomers profoundly alter amino acid metabolism, modulate cytokine/chemokine release and reduce interferon signalling, oxidative stress and the release of viral particles. Of the three isomers, citraconate is the strongest electrophile and nuclear factor-erythroid 2-related factor 2 (NRF2) agonist. Only citraconate inhibits catalysis of itaconate by cis-aconitate decarboxylase (ACOD1), probably by competitive binding to the substrate-binding site. These results reveal mesaconate and citraconate as immunomodulatory, anti-oxidative and antiviral compounds, and citraconate as the first naturally occurring ACOD1 inhibitor.

Identification of the predominant antigenic epitopes in intestinal flora in IBD.

The normal intestinal flora is required for the development of intestinal inflammation in animal models of inflammatory bowel disease (IBD). In humans, several studies indicated a potential association of Escherichia coli (E. coli) with IBD. In addition, we have shown that T-cell clones of IBD patients cross react toward different enteric bacterial species and thus likely respond to conserved bacterial antigens. Therefore, we hypothesized that highly conserved E. coli proteins might be a reasonable candidate to screen for abnormal T-cell responses in IBD. We used high-throughput techniques for cloning, expression, and purification under native conditions of a set of 271 conserved proteins of E. coli, of which 196 were used for whole blood stimulations to assess peripheral T helper (T(H))-cell responses. In addition, because of the association of an adherent-invasive E. coli with Crohn's disease (CD), we included 13 pathogenicity factors of E. coli in the study. We observed that pools of these conserved E. coli proteins less frequently induced interferon-gamma (IFNgamma) production in peripheral T(H) cells in patients with CD and ankylosing spondylitis (AS) compared with healthy controls. In addition, lower percentage of patients with CD and AS responded toward single proteins. The reason for the decreased frequency of an in vitro T(H)-cell IFNgamma response toward E. coli proteins in peripheral blood of CD and AS patients, e.g., increased suppression needs to be clarified.

Recombinant protein expression and solubility screening in Escherichia coli: a comparative study.

Producing soluble proteins in Escherichia coli is still a major bottleneck for structural proteomics. Therefore, screening for soluble expression on a small scale is an attractive way of identifying constructs that are likely to be amenable to structural analysis. A variety of expression-screening methods have been developed within the Structural Proteomics In Europe (SPINE) consortium and to assist the further refinement of such approaches, eight laboratories participating in the network have benchmarked their protocols. For this study, the solubility profiles of a common set of 96 His(6)-tagged proteins were assessed by expression screening in E. coli. The level of soluble expression for each target was scored according to estimated protein yield. By reference to a subset of the proteins, it is demonstrated that the small-scale result can provide a useful indicator of the amount of soluble protein likely to be produced on a large scale (i.e. sufficient for structural studies). In general, there was agreement between the different groups as to which targets were not soluble and which were the most soluble. However, for a large number of the targets there were wide discrepancies in the results reported from the different screening methods, which is correlated with variations in the procedures and the range of parameters explored. Given finite resources, it appears that the question of how to most effectively explore ;expression space' is similar to several other multi-parameter problems faced by crystallographers, such as crystallization.

Protein in gels, computers, crystals and camels.

The IBC's Proteomics 2001 meeting was held in Philadelphia, PA, USA, 14-17 May 2000.

Protein array technology. Potential use in medical diagnostics.

The human genome is sequenced, but only a minority of genes have been assigned a function. Whole-genome expression profiling is an important tool for functional genomic studies. Automated technology allows high-throughput gene activity monitoring by analysis of complex expression patterns, resulting in fingerprints of diseased versus normal or developmentally distinct tissues. Differential gene expression can be most efficiently monitored by DNA hybridization on arrays of oligonucleotides or cDNA clones. Starting from high-density filter membranes, cDNA microarrays have recently been devised in chip format. We have shown that the same cDNA libraries can be used for high-throughput protein expression and antibody screening on high-density filters and microarrays. These libraries connect recombinant proteins to clones identified by DNA hybridization or sequencing, hence creating a direct link between gene catalogs and functional catalogs. Microarrays can now be used to go from an individual clone to a specific gene and its protein product. Clone libraries become amenable to database integration including all steps from DNA sequencing to functional assays of gene products.

Improvements in protein identification by MALDI-TOF-MS peptide mapping.

A new strategy for identifying proteins in sequence data-bases by MALDI-MS peptide mapping is reported. The strategy corrects for systematic deviations of determined peptide molecular masses using information contained in the opened database and thereby renders unnecessary internal spectrum calibration. As a result, data acquisition is simplified and less error prone. Performance of the new strategy is demonstrated by identification of a set of recombinant, human cDNA expression products as well as native proteins isolated from crude mouse brain extracts by 2-D electrophoresis. Using one set of calibration constants for the mass spectrometric analyses, 20 proteins were identified without applying any molecular weight restrictions, which was not possible without data correction. A sequence database search program has been written that performs all necessary calculations automatically, access to which will be provided to the scientific community in the Internet.

By-passing selection: direct screening for antibody-antigen interactions using protein arrays.

We have developed a system to identify highly specific antibody-antigen interactions by protein array screening. This removes the need for selection using animal immunisation or in vitro techniques such as phage or ribosome display. We screened an array of 27 648 human foetal brain proteins with 12 well-expressed antibody fragments that had not previously been exposed to any antigen. Four highly specific antibody-antigen pairs were identified, including three antibodies that bind proteins of unknown function. The target proteins were expressed at a very low copy number on the array, emphasising the unbiased nature of the screen. The specificity and sensitivity of binding demonstrates that this 'naive' screening approach could be applied to the high throughput isolation of specific antibodies against many different targets in the human proteome.

Protein arrays for gene expression and molecular interaction screening.

The array format has revolutionised biomedical experimentation and diagnostics, enabling ordered high-throughput analysis. During the past decade, classic solid phase substrates, such as microtitre plates, membrane filters and microscopic slides, were turned into high-density, chip-like structures. The concept of the arrayed library was central to this development which now extends from DNA to protein. The new and versatile protein array technology allows high-throughput screening for gene expression and molecular interactions. As a major platform for functional genomics, it is already on its way into medical diagnostics.

A human cDNA library for high-throughput protein expression screening.

We have constructed a human fetal brain cDNA library in an Escherichia coli expression vector for high-throughput screening of recombinant human proteins. Using robot technology, the library was arrayed in microtiter plates and gridded onto high-density filter membranes. Putative expression clones were detected on the filters using an antibody against the N-terminal sequence RGS-His(6) of fusion proteins. Positive clones were rearrayed into a new sublibrary, and 96 randomly chosen clones were analyzed. Expression products were analyzed by SDS-PAGE, affinity purification, matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry, and the determined protein masses were compared to masses predicted from DNA sequencing data. It was found that 66% of these clones contained inserts in a correct reading frame. Sixty-four percent of the correct reading frame clones comprised the complete coding sequence of a human protein. High-throughput microtiter plate methods were developed for protein expression, extraction, purification, and mass spectrometric analyses. An enzyme assay for glyceraldehyde-3-phosphate dehydrogenase activity in native extracts was adapted to the microtiter plate format. Our data indicate that high-throughput screening of an arrayed protein expression library is an economical way of generating large numbers of clones producing recombinant human proteins for structural and functional analyses.

Construction and analysis of arrayed cDNA libraries.

For any attempt to understand the biology of an organism the incorporation of a cDNA-based approach is unavoidable, because it is a major approach to studying gene function. The complete sequence of the genome alone is not sufficient to understand any organism; its gene regulation, expression, splice variation, posttranslational modifications, and protein-protein interactions all need to be addressed. Because the majority of vertebrate genes have probably been identified as ESTs the next stage of the Human Genome Project is attributing functional information to these sequences. In most cases hybridization-based approaches on arrayed pieces of DNA represent the most efficient way to study the expression level and splicing of a gene in a given tissue. Similar technology, now being applied at the protein level using protein expression libraries, high-density protein membranes, and antibody screening, should allow studies of protein localization and modifications. Coupled to these approaches is the use of technologies, which although lacking the highly parallel nature of hybridization, can potentially characterize large numbers of samples individually and with high accuracy. Automated gel-based DNA sequencing is an example of such a technique; protein sequencing and mass fingerprinting are further examples. In the case of mass spectroscopic analysis, the speed and sensitivity are vastly superior to that of gel-based approaches; however, the preparation of samples is more tedious. Our laboratory is developing a system to characterize DNA samples by mass spectrometry, allowing more rapid genotyping than is currently possible using gel-based technologies ([symbol: see text]. Gut, [symbol: see text]. Berlin and H. Lehrach, personal communication, 1998). Such technology would make information on gene polymorphisms widely accessible. Data generated using all of these techniques at the DNA and protein level will be connected by both protein expression libraries and database comparisons; finally, two hybrid library screens will identify many of the protein-protein interactions, linking genes together. In this way we will start to understand the interplay between genes on a global scale, both at the level of molecular interaction and the biological processes they regulate.

Protein microarrays for gene expression and antibody screening.

Proteins translate genomic sequence information into function, enabling biological processes. As a complementary approach to gene expression profiling on cDNA microarrays, we have developed a technique for high-throughput gene expression and antibody screening on chip-size protein microarrays. Using a picking/spotting robot equipped with a new transfer stamp, protein solutions were gridded onto polyvinylidene difluoride filters at high density. Specific purified protein was detected on the filters with high sensitivity (250 amol or 10 pg of a test protein). On a microarray made from bacterial lysates of 92 human cDNA clones expressed in a microtiter plate, putative protein expressors could be reliably identified. The rate of false-positive clones, expressing proteins in incorrect reading frames, was low. Product specificity of selected clones was confirmed on identical microarrays using monoclonal antibodies. Cross-reactivities of some antibodies with unrelated proteins imply the use of protein microarrays for antibody specificity screening against whole libraries of proteins. Because this application would not be restricted to antigen-antibody systems, protein microarrays should provide a general resource for high-throughput screens of gene expression and receptor-ligand interactions.

A method for global protein expression and antibody screening on high-density filters of an arrayed cDNA library.

We have developed a technique to establish catalogues of protein products of arrayed cDNA clones identified by DNA hybridisation or sequencing. A human fetal brain cDNA library was directionally cloned in a bacterial vector that allows IPTG-inducible expression of His6-tagged fusion proteins. Using robot technology, the library was arrayed in microtitre plates and gridded onto high-density in situ filters. A monoclonal antibody recognising the N-terminal RGSH6sequence of expressed proteins (RGS.His antibody, Qiagen) detected 20% of the library as putative expression clones. Two example genes, GAPDH and HSP90alpha, were identified on high-density filters using DNA probes and antibodies against their proteins.