Background: Methods are now available for the prediction of interaction sites in protein 3D structures. While many of these methods report high success rates for site prediction, often these predictions are not very selective and have low precision. Precision in site prediction is addressed using Theoretical Microscopic Titration Curves (THEMATICS), a simple computational method for the identification of active sites in enzymes. Recall and precision are measured and compared with other methods for the prediction of catalytic sites.

A new monotonicity-constrained maximum likelihood approach, called Partial Order Optimum Likelihood (POOL), is presented and applied to the problem of functional site prediction in protein 3D structures, an important current challenge in genomics. The input consists of electrostatic and geometric properties derived from the 3D structure of the query protein alone. Sequence-based conservation information, where available, may also be incorporated. Electrostatics features from THEMATICS are combined with multidimensional isotonic regression to form maximum likelihood estimates of probabilities that specific residues belong to an active site. This allows likelihood ranking of all ionizable residues in a given protein based on THEMATICS features. The corresponding ROC curves and statistical significance tests demonstrate that this method outperforms prior THEMATICS-based methods, which in turn have been shown previously to outperform other 3D-structure-based methods for identifying active site residues. Then it is shown that the addition of one simple geometric property, the size rank of the cleft in which a given residue is contained, yields improved performance. Extension of the method to include predictions of non-ionizable residues is achieved through the introduction of environment variables. This extension results in even better performance than THEMATICS alone and constitutes to date the best functional site predictor based on 3D structure only, achieving nearly the same level of performance as methods that use both 3D structure and sequence alignment data. Finally, the method also easily incorporates such sequence alignment data, and when this information is included, the resulting method is shown to outperform the best current methods using any combination of sequence alignments and 3D structures. Included is an analysis demonstrating that when THEMATICS features, cleft size rank, and alignment-based conservation scores are used individually or in combination THEMATICS features represent the single most important component of such classifiers.

Retinal pigment epithelium (RPE) cells are vital for retinal health. However, they are susceptible to injury with ageing and exposure to excessive light, including UV (100-380 nm) and visible (380-760 nm) radiation. To evaluate the protective effect of blueberry anthocyanins on RPE cells, in vitro cell models of replicative senescent and light-induced damage were established in the present study. After purification and fractionation, blueberry anthocyanin extracts (BAE) were yielded with total anthocyanin contents of 31·0 (SD 0·5) % and were used in this study. Replicative senescence of RPE cells was induced by repeatedly passaging cells from the fourth passage to the tenth. From the fifth passage, cultured RPE cells began to enter a replicative senescence, exhibiting reduced cell proliferation along with an increase in the number of β-galactosidase-positive cells. RPE cells maintained high cell viability (P < 0·01) and a low (P < 0·01) percentage of β-galactosidase-positive cells when treated with 0·1 μg/ml BAE. In contrast, after exposure to 2500 (SD 500) lx light (420-800 nm) for 12 h, RPE cells in the positive control (light exposure, no BAE treatment) exhibited premature senescence, low (P < 0·01) cell viability and increased (P < 0·01) vascular endothelial growth factor (VEGF) release compared with negative control cells, which were not subjected to light irradiation and BAE exposure. Correspondingly, BAE is beneficial to RPE cells by protecting these cells against light-induced damage through the suppression of ageing and apoptosis as well as the down-regulation of the over-expressed VEGF to normal level. These results demonstrate that BAE is efficacious against senescence and light-induced damage of RPE cells.

Genomics has posed the challenge of determination of protein function from sequence and/or 3-D structure. Functional assignment from sequence relationships can be misleading, and structural similarity does not necessarily imply functional similarity. Proteins in the DJ-1 family, many of which are of unknown function, are examples of proteins with both sequence and fold similarity that span multiple functional classes. THEMATICS (theoretical microscopic titration curves), an electrostatics-based computational approach to functional site prediction, is used to sort proteins in the DJ-1 family into different functional classes. Active site residues are predicted for the eight distinct DJ-1 proteins with available 3-D structures. Placement of the predicted residues onto a structural alignment for six of these proteins reveals three distinct types of active sites. Each type overlaps only partially with the others, with only one residue in common across all six sets of predicted residues. Human DJ-1 and YajL from Escherichia coli have very similar predicted active sites and belong to the same probable functional group. Protease I, a known cysteine protease from Pyrococcus horikoshii, and PfpI/YhbO from E. coli, a hypothetical protein of unknown function, belong to a separate class. THEMATICS predicts a set of residues that is typical of a cysteine protease for Protease I; the prediction for PfpI/YhbO bears some similarity. YDR533Cp from Saccharomyces cerevisiae, of unknown function, and the known chaperone Hsp31 from E. coli constitute a third group with nearly identical predicted active sites. While the first four proteins have predicted active sites at dimer interfaces, YDR533Cp and Hsp31 both have predicted sites contained within each subunit. Although YDR533Cp and Hsp31 form different dimers with different orientations between the subunits, the predicted active sites are superimposable within the monomer structures. Thus, the three predicted functional classes form four different types of quaternary structures. The computational prediction of the functional sites for protein structures of unknown function provides valuable clues for functional classification.

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