Uniformly curated signaling pathways reveal tissue-specific cross-talks and support drug target discovery.

MOTIVATION: Signaling pathways control a large variety of cellular processes. However, currently, even within the same database signaling pathways are often curated at different levels of detail. This makes comparative and cross-talk analyses difficult. RESULTS: We present SignaLink, a database containing eight major signaling pathways from Caenorhabditis elegans, Drosophila melanogaster and humans. Based on 170 review and approximately 800 research articles, we have compiled pathways with semi-automatic searches and uniform, well-documented curation rules. We found that in humans any two of the eight pathways can cross-talk. We quantified the possible tissue- and cancer-specific activity of cross-talks and found pathway-specific expression profiles. In addition, we identified 327 proteins relevant for drug target discovery. CONCLUSIONS: We provide a novel resource for comparative and cross-talk analyses of signaling pathways. The identified multi-pathway and tissue-specific cross-talks contribute to the understanding of the signaling complexity in health and disease, and underscore its importance in network-based drug target selection. AVAILABILITY: http://SignaLink.org.

The small heat-shock proteins HSPB2 and HSPB3 form well-defined heterooligomers in a unique 3 to 1 subunit ratio.

Various mammalian small heat-shock proteins (sHSPs) can interact with one another to form large polydisperse assemblies. In muscle cells, HSPB2/MKBP (myotonic dystrophy protein kinase-binding protein) and HSPB3 have been shown to form an independent complex. To date, the biochemical properties of this complex have not been thoroughly characterized. In this study, we show that recombinant HSPB2 and HSPB3 can be successfully purified from Escherichia coli cells co-expressing both proteins. Nanoelectrospray ionization mass spectrometry and sedimentation velocity analytical ultracentrifugation analysis showed that HSPB2/B3 forms a series of well defined hetero-oligomers, consisting of 4, 8, 12, 16, 20 and 24 subunits, each maintaining a strict 3:1 HSPB2/HSPB3 subunit ratio. These complexes are thermally stable up to 40 degrees C, as determined by far-UV circular dichroism spectroscopy. Surprisingly, HSPB2/B3 exerted a poor chaperone-like and thermoprotective activity, which is likely related to the low surface hydrophobicity, as revealed by its interaction with the hydrophobic probe 1-anilino-8-naphthalenesulfonic acid. Co-immunoprecipitation experiments demonstrated that the HSPB2/B3 oligomer cannot interact with HSP20, HSP27 or alphaB-crystallin, whereas the homomeric form of HSPB2, thus not in complex with HSPB3, could associate efficiently with HSP20. Taken altogether, this study provides evidence that, despite the high level of sequence homology within the sHSP family the biochemical properties of the HSPB2/B3 complex are distinctly different from those of other sHSPs, indicating that the HSPB2/B3 assembly is likely to possess cellular functions other than those of its family members.

Perturbation waves in proteins and protein networks: applications of percolation and game theories in signaling and drug design.

The network paradigm is increasingly used to describe the dynamics of complex systems. Here we review the current results and propose future development areas in the assessment of perturbation waves, i.e. propagating structural changes in amino acid networks building individual protein molecules and in protein-protein interaction networks (interactomes). We assess the possibilities and critically review the initial attempts for the application of game theory to the often rather complicated process, when two protein molecules approach each other, mutually adjust their conformations via multiple communication steps and finally, bind to each other. We also summarize available data on the application of percolation theory for the prediction of amino acid network- and interactome-dynamics. Furthermore, we give an overview of the dissection of signals and noise in the cellular context of various perturbations. Finally, we propose possible applications of the reviewed methodologies in drug design.

Network analysis of protein dynamics.

The network paradigm is increasingly used to describe the topology and dynamics of complex systems. Here, we review the results of the topological analysis of protein structures as molecular networks describing their small-world character, and the role of hubs and central network elements in governing enzyme activity, allosteric regulation, protein motor function, signal transduction and protein stability. We summarize available data how central network elements are enriched in active centers and ligand binding sites directing the dynamics of the entire protein. We assess the feasibility of conformational and energy networks to simplify the vast complexity of rugged energy landscapes and to predict protein folding and dynamics. Finally, we suggest that modular analysis, novel centrality measures, hierarchical representation of networks and the analysis of network dynamics will soon lead to an expansion of this field.

Stress-induced rearrangements of cellular networks: Consequences for protection and drug design.

The complexity of the cells can be described and understood by a number of networks such as protein-protein interaction, cytoskeletal, organelle, signalling, gene transcription and metabolic networks. All these networks are highly dynamic producing continuous rearrangements in their links, hubs, network-skeleton and modules. Here we describe the adaptation of cellular networks after various forms of stress causing perturbations, congestions and network damage. Chronic stress decreases link-density, decouples or even quarantines modules, and induces an increased competition between network hubs and bridges. Extremely long or strong stress may induce a topological phase transition in the respective cellular networks, which switches the cell to a completely different mode of cellular function. We summarize our initial knowledge on network restoration after stress including the role of molecular chaperones in this process. Finally, we discuss the implications of stress-induced network rearrangements in diseases and ageing, and propose therapeutic approaches both to increase the robustness and help the repair of cellular networks.

How to design multi-target drugs.

Despite improved rational drug design and a remarkable progress in genomic, proteomic and high-throughput screening methods, the number of novel, single-target drugs has fallen far behind expectations during the past decade. Multi-target drugs multiply the number of pharmacologically relevant target molecules by introducing a set of indirect, network-dependent effects. Parallel with this, the low-affinity binding of multi-target drugs eases the constraints of druggability and significantly increases the size of the druggable proteome. These effects tremendously expand the number of potential drug targets and introduce novel classes of multi-target drugs with smaller side effects and toxicity. Here, the authors review the recent progress in this field, compare possible network attack strategies and propose several methods to find target-sets for multi-target drugs.

Temperature effects on alpha-crystallin structure probed by 6-bromomethyl-2-(2-furanyl)-3-hydroxychromone, an environmentally sensitive two-wavelength fluorescent dye covalently attached to the single Cys residue.

The single Cys residue in the C-terminal domain of bovine eye lens alpha-crystallin was covalently labelled with 6-bromomethyl-2-(2-furanyl)-3-hydroxychromone. This novel SH-reactive two-band ratiometric fluorescent dye is characterized by excited state intramolecular proton transfer reaction yielding two highly emissive N* and T* bands separated by more than 100 nm. Their relative intensities are known to be highly sensitive to the H-bonding ability of the environment. Properties of the environment of the dye attached to the protein were studied under native-like conditions and at a range of elevated temperatures that are known to facilitate alpha-crystallin chaperone-like activity. We observe that on heating, the environment of the dye becomes more flexible and the H-bonding of the dye with the protein vicinity decreases. The spectroscopic properties observed on heating were partially restored after cooling, but the initial values were not reached on the time scale of our experiments (up to 3 h). This suggests that the changes of the dye microenvironment are connected with the rearrangements of alpha-crystallin quaternary structure. Since there is only one Cys residue in alphaA subunit of alpha-crystallin (whereas alphaB subunit contains no Cys), we attributed the observed temperature-induced changes of the dye's microenvironment to the particular site within alpha-crystallin molecule.

Chaperone-like activity of alpha-crystallin is enhanced by high-pressure treatment.

alpha-Crystallin, an oligomeric protein in vertebrate eye lens, is a member of the small heat-shock protein family. Several papers pointed out that its chaperone-like activity could be enhanced by increasing the temperature. We demonstrate in the present study that structural perturbations by high hydrostatic pressures up to 300 MPa also enhance this activity. In contrast with temperature-induced changes, the pressure-induced enhancement is reversible. After pressure release, the extra activity is lost with a relaxation time of 2.0+/-0.5 h. Structural alterations contributing to the higher activity were studied with IR and fluorescence spectroscopy, and light-scattering measurements. The results suggest that while the secondary structure barely changes under pressure, the interactions between the subunits weaken, the oligomers dissociate, the area of accessible hydrophobic surfaces significantly increases and the environment of tryptophan residues becomes slightly more polar. It seems that structural flexibility and the total surface area of the oligomers are the key factors in the chaperone capacity, and that the increase in the chaperone activity does not require the increase in the oligomer size as was assumed previously [Burgio, Kim, Dow and Koretz (2000) Biochem. Biophys. Res. Commun. 268, 426-432]. After pressure release, the structure of subunits are reorganized relatively quickly, whereas the oligomer size reaches its original value slowly with a relaxation time of 33+/-4 h. In our interpretation, both the fast and slow structural rearrangements have an impact on the functional relaxation.