Systems biology approaches to enhance our understanding of drug hypersensitivity reactions.

Hypersensitivity drug reactions (HDRs) encompass a wide spectrum of unpredictable clinical entities. They represent an important health problem, affecting people of all ages, and lead to a large strain on the public health system. Here, we summarize experiments that use high-throughput genomics technologies to investigate HDRs. We also introduce the field of systems biology as a relatively recent discipline concerned with the integration and analysis of high-throughput data sets such as DNA microarrays and next-generation sequencing data. We describe previous studies that have applied systems biology techniques to related fields such as allergy and asthma. Finally, we present a number of potential applications of systems biology to the study of HDRs, in order to make the reader aware of the types of analyses that can be performed and the insights that can be gained through their application.

Regulatory cross-talk of mouse liver polyamine and methionine metabolic pathways: a systemic approach to its physiopathological consequences.

Both polyamines and methionine derivatives are nitrogen compounds directly related to the regulation of gene expression. In silico predictions and experimental evidence suggest a cross-talk between polyamine and methionine metabolism in mammalian tissues. Since liver is the major organ that controls nitrogen metabolism of the whole organism, it is the best tissue to further test this hypothesis in vivo. In this work, we studied the effects of the chronic administration of a methionine-supplemented diet (0.5% Met in drinking water for 5 months) on the liver of mice (designated as MET-mice). Metabolic and proteomic approaches were performed and the data obtained were subjected to biocomputational analysis. Results showed that a supplemental methionine intake can indeed regulate biogenic amine metabolism in an in vivo model by multiple mechanisms including metabolic regulation and specific gene demethylation. Furthermore, putative systemic effects were investigated by molecular and cellular biology methods. Among other results, altered expression levels of multiple inflammation and cell proliferation/death balance markers were found and macrophage activation was observed. Overall, the results presented here will be of interest across a variety of biomedical disciplines, including nutrition, orphan diseases, immunology and oncology.

Systematic computational prediction of protein interaction networks.

Determining the network of physical protein associations is an important first step in developing mechanistic evidence for elucidating biological pathways. Despite rapid advances in the field of high throughput experiments to determine protein interactions, the majority of associations remain unknown. Here we describe computational methods for significantly expanding protein association networks. We describe methods for integrating multiple independent sources of evidence to obtain higher quality predictions and we compare the major publicly available resources available for experimentalists to use.

Model of the ran-RCC1 interaction using biochemical and docking experiments.

RCC1, the regulator of chromosome condensation, is the guanine nucleotide exchange factor (GEF) for the nuclear Ras-like GTP-binding protein Ran. Its structure was solved by X-ray crystallography and revealed a seven-bladed beta-propeller, one side of which was proposed to be the interaction site with Ran. To gain more insight into this interaction, alanine mutagenesis studies were performed on conserved residues on the surface of the structure. Purified mutant proteins were analysed by steady-state kinetic analysis of their GEF activities towards Ran. A number of residues were identified whose mutation affected either the KMor kcatof the overall reaction, or had no effect. Mutants were further analysed by plasmon surface resonance in order to get more information on individual steps of the complex reaction pathway. Ran-GDP was coupled to the sensor chip and reacted with RCC1 mutants to categorise them into different groups, demonstrating the usefulness of plasmon surface resonance in the study of complex multi-step kinetic processes. A docking solution of Ran-RCC1 structures in combination with sequence analysis allows prediction of the site of interaction between RCC1 and Ran and proposes a model for the Ran-RCC1 structure which corresponds to and extends the biochemical data. Three invariant residues which most severely affect the kcatof the reaction, D128, D182 and H304, are located in the centre of the Ran-RCC1 interface and interfere with switch II and the phosphate binding area. The structural model suggests that different guanine nucleotide exchange factors use a similar interaction site on their respective GTP-binding proteins, but that the molecular mechanisms for the release of nucleotides are likely to be different.

Distribution and functional diversification of the ras superfamily in Saccharomyces cerevisiae.

The recent availability of the full Saccharomyces cerevisiae genome sequence offers a first opportunity to analyze the composition, function and evolution of GTPases in the ras-p21 superfamily. This superfamily in yeast is composed of 29 proteins divided into five families: ras with four sequences implicated in cell signalling; rho, six genes related to the cell shape machinery; ypt-rab, ten proteins with different roles in intracellular trafficking; arf-sar, seven proteins related to vesicular trafficking in secretory pathways; and ran, two proteins acting as components of the nuclear transport system. The superfamily covers a wide range of cellular functions from signalling to intracellular trafficking, while conserving the structural framework and a common mechanism of GTP hydrolysis.

p23 and HSP20/alpha-crystallin proteins define a conserved sequence domain present in other eukaryotic protein families.

We identified families of proteins characterized by the presence of a domain similar to human p23 protein, which include proteins such as Sgt1, involved in the yeast kinetochore assembly; melusin, involved in specific interactions with the cytoplasmic integrin beta1 domain; Rar1, related to pathogenic resistance in plants, and to development in animals; B5+B5R flavo-hemo cytochrome NAD(P)H oxidoreductase type B in humans and mice; and NudC, involved in nucleus migration during mitosis. We also found that p23 and the HSP20/alpha-crystallin family of heat shock proteins, which share the same three-dimensional folding, show a pattern of conserved residues that points to a common origin in the evolution of both protein domains. The p23 and HSP20/alpha-crystallin phylogenetic relationship and their similar role in chaperone activity suggest a common function, probably involving protein-protein interaction, for those proteins containing p23-like domains.

Search for ancient patterns in protein sequences.

Proteins of related functions are often similar in sequence, reflecting a common phylogenetic origin. Proteins with no known homology are probably diversified proteins, too distantly related to known sequences in databases to retain significant similarity. All proteins, however, probably share common ancestries if one moves far enough back in evolution; therefore, given the huge accumulation of protein sequences in current databases, it could be expected that some proteins with no obvious sequence resemblance to any other share some residues that could represent footprints of ancient common ancestries. To identify such putative footprints, we have searched for short stretches of amino acids present in a given protein sequence that are also found in a significant number of nonrelated proteins in the database. The significantly high frequency of occurrence of these "patterns" in the database would support a common evolutionary source, and a diversity of non-related proteins that contain the pattern would express their ancient origin. Using this strategy, significant patterns were found in actual exons, but not in randomized amino acid sequences, nor in "translated" sequences of noncoding DNA, suggesting that this strategy actually leads to the identification of patterns with a biological significance. These significant patterns are not randomly positioned along the sequences analyzed, but they tend to accumulate within specific regions, producing a profile of discrete "domains." In some well-known proteins analyzed in this study, some of these domains are coincident with known motifs. Thus, the procedure described in this paper could be useful for identifying ancient patterns and domains in protein sequences, some of which could also have a functional or structural significance.

Immune recognition of swine vesicular disease virus structural proteins: novel antigenic regions that are not exposed in the capsid.

Swine vesicular disease virus (SVDV) is an enterovirus of the Picornaviridae family that belongs to the coxsackievirus B group. A number of antigenic sites have been identified in SVDV by analysis of neutralizing monoclonal antibody-resistant mutants and shown to be exposed on the surface of the capsid. In this paper we have identified seven new immunodominant antigenic regions in SVDV capsid proteins by a peptide scanning method, using a panel of sera from infected pigs. When these antigenic regions were located in the capsid by using a computer-generated three-dimensional model of the virion, one was readily exposed on the surface of the virus and the remaining sites were located facing the inner side of the capsid shell, at subunit contacts, or in the interior of the subunit structure.

Effector recognition by the small GTP-binding proteins Ras and Ral.

The Ral effector protein RLIP76 (also called RIP/RalBP1) binds to Ral.GTP via a region that shares no sequence homology with the Ras-binding domains of the Ser/Thr kinase c-Raf-1 and the Ral-specific guanine nucleotide exchange factors. Whereas the Ras-binding domains have a similar ubiquitin-like structure, the Ral-binding domain of RLIP was predicted to comprise a coiled-coil region. In order to obtain more information about the specificity and the structural mode of the interaction between Ral and RLIP, we have performed a sequence space and a mutational analysis. The sequence space analysis of a comprehensive nonredundant assembly of Ras-like proteins strongly indicated that positions 36 and 37 in the core of the effector region are tree-determinant positions for all subfamilies of Ras-like proteins and dictate the specificity of the interaction of these GTPases with their effector proteins. Indeed, we could convert the specific interaction with Ras effectors and RLIP by mutating these residues in Ras and Ral. We therefore conclude that positions 36 and 37 are critical for the discrimination between Ras and Ral effectors and that, despite the absence of sequence homology between the Ral-binding and the Ras-binding domains, their mode of interaction is most probably similar.