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Epithelial-to-mesenchymal transition (EMT), a switch of polarized epithelial cells to a migratory, fibroblastoid phenotype, is increasingly considered as an important event during malignant tumor progression and metastasis. To identify molecular players involved in EMT and metastasis, we performed expression profiling of a set of combined in vitro/in vivo cellular models, based on clonal, fully polarized mammary epithelial cells. Seven closely related cell pairs were used, which were modified by defined oncogenes and/or external factors and showed specific aspects of epithelial plasticity relevant to cell migration, local invasion and metastasis. Since mRNA levels do not necessarily reflect protein levels in cells, we used an improved expression profiling method based on polysome-bound RNA, suitable to analyse global gene expression on Affymetrix chips. A substantial fraction of all regulated genes was found to be exclusively controlled at the translational level. Furthermore, profiling of the above multiple cell pairs allowed one to identify small numbers of genes by cluster analysis, specifically correlating gene expression with EMT, metastasis, scattering and/or oncogene function. A small set of genes specifically regulated during EMT was identified, including key regulators and signaling pathways involved in cell proliferation, epithelial polarity, survival and transdifferentiation to mesenchymal-like cells with invasive behavior.

Understanding the mechanism and specificity of substrate binding in the cytochrome P450 (P450) superfamily is an important step toward explaining its key role in drug metabolism, toxicity, xenobiotic degradation, and several biosynthetic pathways. Here we investigate the ligand exit pathways and mechanisms of P450cam (CYP101), P450BM-3 (CYP102), and P450eryF (CYP107A1) by using random expulsion molecular dynamics and classical molecular dynamics simulations. Although several different pathways are found for each protein, one pathway is common to all three. The mechanism of ligand exit along this pathway is, however, quite different in the three different proteins. For P450cam, small backbone conformational changes, in combination with aromatic side chain rotation, allow for the passage of the rather rigid, compact, and hydrophobic substrate, camphor. In P450BM-3, larger transient backbone changes are observed on ligand exit. R47, situated at the entrance to the channel, appears important in guiding negatively charged fatty acid substrates in and out of the active site. In P450eryF, an isolated buried arginine, R185, stabilized by four hydrogen bonds to backbone carbonyl oxygen atoms, is located in the exit channel and is identified as having a particularly unusual functionality, dynamically gating channel opening. The results for these three P450s suggest that the channel opening mechanisms are adjusted to the physico-chemical properties of the substrate and can kinetically modulate protein-substrate specificity.

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To bind at an enzyme's active site, a ligand must diffuse or be transported to the enzyme's surface, and, if the binding site is buried, the ligand must diffuse through the protein to reach it. Although the driving force for ligand binding is often ascribed to the hydrophobic effect, electrostatic interactions also inf luence the binding process of both charged and nonpolar ligands. First, electrostatic steering of charged substrates into enzyme active sites is discussed. This is of particular relevance for diffusion-inf luenced enzymes. By comparing the results of Brownian dynamics simulations and electrostatic potential similarity analysis for triose-phosphate isomerases, superoxide dismutases, and ␤-lactamases from different species, we identify the conserved features responsible for the electrostatic substrate-steering fields. The conserved potentials are localized at the active sites and are the primary determinants of the bimolecular association rates. Then we focus on a more subtle effect, which we will refer to as ''ionic tethering.'' We explore, by means of molecular and Brownian dynamics simulations and electrostatic continuum calculations, how salt links can act as tethers between structural elements of an enzyme that undergo conformational change upon substrate binding, and thereby regulate or modulate substrate binding. This is illustrated for the lipase and cytochrome P450 enzymes. Ionic tethering can provide a control mechanism for substrate binding that is sensitive to the electrostatic properties of the enzyme's surroundings even when the substrate is nonpolar.

The temperature dependence of hydrophobic interactions of methane-like particles in water is analyzed in terms of free energy, entropy, internal energy, and the second osmotic virial coefficient. A large computational effort (approximately 15 ns cumulative trajectory length at each temperature) has been undertaken in order to guarantee reliable free energy and entropy data. At 300 K association is controlled by entropy, but as the temperature rises the internal energy takes over and dominates at 500 K. Both internal energy and entropy change sign within this temperature range. Our results correspond qualitatively with the experimentally observed temperature effect for transfer of gaseous hydrophobic substances into water:  ΔA shows a weak temperature dependence, while ΔE and ΔS vary strongly with temperature. The second osmotic virial coefficients were calculated at different temperatures. Agreement with osmotic virial coefficients measured by solubility experiments at 300 K was found. Our results indicate that pairwise hydrophobic association studied by molecular dynamics simulation shows the key effects reported for bulk hydrophobic interactions. At present, there is no evidence for a qualitative difference between pair and bulk hydrophobic interactions. It is demonstrated that the comparison of the second osmotic virial coefficient of the solute particles in water, B 2,aq, with that in the pure gas phase, B 2,g, is not apppropriate for an assessment of the influence of water on pairwise hydrophobic interactions.

The association of a pair of hydrophobic solutes in water has been investigated by free energy molecular dynamics simulations of a system containing 516 water molecules. Convergence of the calculations is guaranteed by the comparison of data obtained with two independent free energy sampling techniques, which have been optimized for our system. Coulomb interactions have been treated with the Ewald method. Using this computationally expensive approach many of the previously reported discrepancies in the temperature, pressure and interaction parameter dependence of hydrophobic association are clarified. A temperature effect on both the free energy of association and the equilibrium between contact and solvent-separated species is observed. Raising temperature favors association. The most pronounced temperature dependence occurs in the interval between 300 and 350K.

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