Purification and Characterization of Native and Vaccine Candidate Mutant Enterotoxigenic Escherichia coli Heat-Stable Toxins.

Enterotoxigenic Escherichia coli (ETEC), which secretes the heat-stable toxin (ST) is among the four most important enteropathogens that cause moderate-to-severe diarrhea in children in low- and middle-income countries. ST is an intestinal molecular antagonist causing diarrhea and hence an attractive vaccine target. A non-toxic and safe ST vaccine should include one or more detoxifying mutations, and rigorous characterization of such mutants requires structurally intact peptides. To this end, we established a system for purification of ST and ST mutants by fusing the sequence encoding the mature ST peptide to the disulfide isomerase DsbC. A Tobacco Etch Virus protease cleavage site facilitates the proteolytic release of free ST with no additional residues. The purified ST peptides have the expected molecular masses, the correct number of disulfide bridges, and have biological activities and antigenic properties comparable to ST isolated from ETEC. We also show that free DsbC can assist in refolding denatured and misfolded ST in vitro. Finally, we demonstrate that the purification system can be used to produce ST mutants with an intact neutralizing epitope, that two single mutations, L9S and A14T, reduce toxicity more than 100-fold, and that the L9S/A14T double mutant has no measurable residual toxicity.

Mutational analysis of the pro-peptide of a marine intracellular subtilisin protease supports its role in inhibition.

Intracellular subtilisin proteases (ISPs) have important roles in protein processing during the stationary phase in bacteria. Their unregulated protein degrading activity may have adverse effects inside a cell, but little is known about their regulatory mechanism. Until now, ISPs have mostly been described from Bacillus species, with structural data from a single homolog. Here, we study a marine ISP originating from a phylogenetically distinct genus, Planococcus sp. The enzyme was successfully overexpressed in E. coli, and is active in presence of calcium, which is thought to have a role in minor, but essential, structural rearrangements needed for catalytic activity. The ISP operates at alkaline pH and at moderate temperatures, and has a corresponding melting temperature around 60 °C. The high-resolution 3-dimensional structure reported here, represents an ISP with an intact catalytic triad albeit in a configuration with an inhibitory pro-peptide bound. The pro-peptide is removed in other homologs, but the removal of the pro-peptide from the Planococcus sp. AW02J18 ISP appears to be different, and possibly involves several steps. A first processing step is described here as the removal of 2 immediate N-terminal residues. Furthermore, the pro-peptide contains a conserved LIPY/F-motif, which was found to be involved in inhibition of the catalytic activity.

SBMLmod: a Python-based web application and web service for efficient data integration and model simulation.

BACKGROUND: Systems Biology Markup Language (SBML) is the standard model representation and description language in systems biology. Enriching and analysing systems biology models by integrating the multitude of available data, increases the predictive power of these models. This may be a daunting task, which commonly requires bioinformatic competence and scripting. RESULTS: We present SBMLmod, a Python-based web application and service, that automates integration of high throughput data into SBML models. Subsequent steady state analysis is readily accessible via the web service COPASIWS. We illustrate the utility of SBMLmod by integrating gene expression data from different healthy tissues as well as from a cancer dataset into a previously published model of mammalian tryptophan metabolism. CONCLUSION: SBMLmod is a user-friendly platform for model modification and simulation. The web application is available at http://sbmlmod.uit.no , whereas the WSDL definition file for the web service is accessible via http://sbmlmod.uit.no/SBMLmod.wsdl . Furthermore, the entire package can be downloaded from https://github.com/MolecularBioinformatics/sbml-mod-ws . We envision that SBMLmod will make automated model modification and simulation available to a broader research community.

Towards Rational Design of a Toxoid Vaccine against the Heat-Stable Toxin of Escherichia coli.

Enterotoxigenic Escherichia coli(ETEC) is an important cause of diarrheal disease and death in children <5 years old. ETEC strains that express the heat-stable toxin (ST), with or without the heat-labile toxin, are among the four most important diarrhea-causing pathogens. This makes ST an attractive target for an ETEC vaccine. An ST vaccine should be nontoxic and elicit an immune response that neutralizes native ST without cross-reacting with the human endogenous guanylate cyclase C receptor ligands. To identify variants of ST with no or low toxicity, we screened a library of all 361 possible single-amino-acid mutant forms of ST by using the T84 cell assay. Moreover, we identified mutant variants with intact epitopes by screening for the ability to bind neutralizing anti-ST antibodies. ST mutant forms with no or low toxicity and intact epitopes are termed toxoid candidates, and the top 30 candidates all had mutations of residues A14, N12, and L9. The identification of nontoxic variants of L9 strongly suggests that it is a novel receptor-interacting residue, in addition to the previously identified N12, P13, and A14 residues. The screens also allowed us to map the epitopes of three neutralizing monoclonal antibodies, one of which cross-reacts with the human ligand uroguanylin. The common dominant epitope residue for all non-cross-reacting antibodies was Y19. Our results suggest that it should be possible to rationally design ST toxoids that elicit neutralizing immune responses against ST with minimal risk of immunological cross-reactivity.

Model of tryptophan metabolism, readily scalable using tissue-specific gene expression data.

Tryptophan is utilized in various metabolic routes including protein synthesis, serotonin, and melatonin synthesis and the kynurenine pathway. Perturbations in these pathways have been associated with neurodegenerative diseases and cancer. Here we present a comprehensive kinetic model of the complex network of human tryptophan metabolism based upon existing kinetic data for all enzymatic conversions and transporters. By integrating tissue-specific expression data, modeling tryptophan metabolism in liver and brain returned intermediate metabolite concentrations in the physiological range. Sensitivity and metabolic control analyses identified expected key enzymes to govern fluxes in the branches of the network. Combining tissue-specific models revealed a considerable impact of the kynurenine pathway in liver on the concentrations of neuroactive derivatives in the brain. Moreover, using expression data from a cancer study predicted metabolite changes that resembled the experimental observations. We conclude that the combination of the kinetic model with expression data represents a powerful diagnostic tool to predict alterations in tryptophan metabolism. The model is readily scalable to include more tissues, thereby enabling assessment of organismal tryptophan metabolism in health and disease.

NAD(+) biosynthesis and salvage--a phylogenetic perspective.

NAD is best known as an electron carrier and a cosubstrate of various redox reactions. However, over the past 20 years, NAD(+) has been shown to be a key signaling molecule that mediates post-translational protein modifications and serves as precursor of ADP-ribose-containing messenger molecules, which are involved in calcium mobilization. In contrast to its role as a redox carrier, NAD(+)-dependent signaling processes involve the release of nicotinamide (Nam) and require constant replenishment of cellular NAD(+) pools. So far, very little is known about the evolution of NAD(P) synthesis in eukaryotes. In the present study, genes involved in NAD(P) metabolism in 45 species were identified and analyzed with regard to similarities and differences in NAD(P) synthesis. The results show that the Preiss-Handler pathway and NAD(+) kinase are present in all organisms investigated, and thus seem to be ancestral routes. Additionally, two pathways exist that convert Nam to NAD(+); we identified several species that have apparently functional copies of both biosynthetic routes, which have been thought to be mutually exclusive. Furthermore, our findings suggest the parallel phylogenetic appearance of Nam N-methyltransferase, Nam phosphoribosyl transferase, and poly-ADP-ribosyltransferases.

Untangling the intracellular signalling network in cancer--a strategy for data integration in acute myeloid leukaemia.

Protein and gene networks centred on the regulatory tumour suppressor proteins may be of crucial importance both in carcinogenesis and in the response to chemotherapy. Tumour suppressor protein p53 integrates intracellular data in stress responses, receiving signals and translating these into differential gene expression. Interpretation of the data integrated on p53 may therefore reveal the response to therapy in cancer. Proteomics offers more specific data - closer to "the real action" - than the hitherto more frequently used gene expression profiling. Integrated data analysis may reveal pathways disrupted at several regulatory levels. Ultimately, integrated data analysis may also contribute to finding key underlying cancer genes. We here proposes a Partial Least Squares Regression (PLSR)-based data integration strategy, which allows simultaneous analysis of proteomic data, gene expression data and classical clinical parameters. PLSR collapses multidimensional data into fewer relevant dimensions for data interpretation. PLSR can also aid identification of functionally important modules by also performing comparison to databases on known biological interactions. Further, PLSR allows meaningful visualization of complex datasets, aiding interpretation of the underlying biology. Extracting the true biological causal mechanisms from heterogeneous patient populations is the key to discovery of new therapeutic options in cancer.

Identification and characterization of retinoblastoma gene mutations disturbing apoptosis in human breast cancers.

BACKGROUND: The tumor suppressor pRb plays a key role regulating cell cycle arrest, and disturbances in the RB1 gene have been reported in different cancer forms. However, the literature reports contradictory findings with respect to a pro--versus anti--apoptotic role of pRb, and the consequence of alterations in RB1 to chemotherapy sensitivity remains unclear. This study is part of a project investigating alterations in pivotal genes as predictive factors to chemotherapy sensitivity in breast cancer. RESULTS: Analyzing 73 locally advanced (stage III) breast cancers, we identified two somatic and one germline single nucleotide changes, each leading to amino acid substitution in the pRb protein (Leu607Ile, Arg698Trp, and Arg621Cys, respectively). This is the first study reporting point mutations affecting RB1 in breast cancer tissue. In addition, MLPA analysis revealed two large multiexon deletions (exons 13 to 27 and exons 21 to 23) with the exons 21-23 deletion occurring in the tumor also harboring the Leu607Ile mutation. Interestingly, Leu607Ile and Arg621Cys point mutations both localize to the spacer region of the pRb protein, a region previously shown to harbor somatic and germline mutations. Multiple sequence alignment across species indicates the spacer to be evolutionary conserved. All three RB1 point mutations encoded nuclear proteins with impaired ability to induce apoptosis compared to wild-type pRb in vitro. Notably, three out of four tumors harboring RB1 mutations displayed primary resistance to treatment with either 5-FU/mitomycin or doxorubicin while only 14 out of 64 tumors without mutations were resistant (p = 0.046). CONCLUSIONS: Although rare, our findings suggest RB1 mutations to be of pathological importance potentially affecting sensitivity to mitomycin/anthracycline treatment in breast cancer.