Proteasome-catalyzed peptide splicing (PCPS) represents an additional activity of mammalian 20S proteasomes recently identified in connection with antigen presentation. We show here that PCPS is not restricted to mammalians but that it is also a feature of yeast 20S proteasomes catalyzed by all three active site  subunits. No major differences in splicing efficiency exist between human 20S standard-and immuno-proteasome or yeast 20S proteasome. Using H 2 18 O to monitor the splicing reaction we also demonstrate that PCPS occurs via direct transpeptidation that slightly favors the generation of peptides spliced in cis over peptides spliced in trans. The 20S proteasome with its proteolytically active site -subunits (1, 2, and 5) is a N-terminal nucleophilic hydrolase, widely conserved during evolution from yeast to mammals. It is the central proteolytic machinery of the ubiquitin proteasome system and the catalytic core of the 26S proteasome that is built by the association of 19S regulator complexes with the 20S proteasome. As part of the 26S proteasome the 20S core degrades poly-ubiquitylated proteins to peptides of 3 to 20 residues in length (1). A small percentage of these peptides is transported to the endoplasmic reticulum, bound by major histocompatibility complex (MHC) 1 class I molecules, and presented at the cell surface to CD8ϩ cytotoxic T lymphocyte for immune recognition. This antigen presentation pathway is usually restricted to the proteasome-dependent processing of self-and viral-proteins (2). Antigen presentation is generally increased after IFN-␥ stimuli because it induces, among others, the synthesis of alternative catalytic subunits (1i, 2i, and 5i) and the concomitant formation of immunoproteasomes (i-proteasomes) (2).All active  subunits carry an N-terminal threonine residue as reactive nucleophile. Therefore, their distinct cleavage preferences are determined by the structural features of the substrate binding pockets. In particular, the nonprimed substrate binding site of the active site  subunits binds the residues of the peptide substrate that are located at the N-terminal side of the cleaved residue. The residues of the peptide located C-terminally of the cleavage site are bound by the primed substrate binding site. The binding to both substrate binding sites of the active site  subunit provides the stability and the orientation of the substrate, which is mandatory to carry out the proteolytic cleavage (3).Peptides can be produced by proteasomes during the degradation of proteins or polypeptides by conventional peptide bond hydrolysis or by proteasome-catalyzed peptide splicing (PCPS). The latter has been demonstrated in vivo so far only for four MHC class I-restricted epitopes (4 -8), leading to the assumption that PCPS is most likely a rare event that lacks any wider functional importance (9). PCPS was suggested to occur in a direct transpeptidation reaction, in either cis or trans, by linking two proteasomal cleavage products (PCPs) derived either from the same or from two ...
The SuperPred web server connects chemical similarity of drug-like compounds with molecular targets and the therapeutic approach based on the similar property principle. Since the first release of this server, the number of known compound–target interactions has increased from 7000 to 665 000, which allows not only a better prediction quality but also the estimation of a confidence. Apart from the addition of quantitative binding data and the statistical consideration of the similarity distribution in all drug classes, new approaches were implemented to improve the target prediction. The 3D similarity as well as the occurrence of fragments and the concordance of physico-chemical properties is also taken into account. In addition, the effect of different fingerprints on the prediction was examined. The retrospective prediction of a drug class (ATC code of the WHO) allows the evaluation of methods and descriptors for a well-characterized set of approved drugs. The prediction is improved by 7.5% to a total accuracy of 75.1%. For query compounds with sufficient structural similarity, the web server allows prognoses about the medical indication area of novel compounds and to find new leads for known targets. SuperPred is publicly available without registration at: http://prediction.charite.de.
One of the most important and challenging tasks in protein modelling is the prediction of loops, as can be seen in the large variety of existing approaches. Loops In Proteins (LIP) is a database that includes all protein segments of a length up to 15 residues contained in the Protein Data Bank (PDB). In this study, the applicability of LIP to loop prediction in the framework of homology modelling is investigated. Searching the database for loop candidates takes less than 1 s on a desktop PC, and ranking them takes a few minutes. This is an order of magnitude faster than most existing procedures. The measure of accuracy is the root mean square deviation (RMSD) with respect to the main-chain atoms after local superposition of target loop and predicted loop. Loops of up to nine residues length were modelled with a local RMSD <1 A and those of length up to 14 residues with an accuracy better than 2 A. The results were compared in detail with a thoroughly evaluated and tested ab initio method published recently and additionally with two further methods for a small loop test set. The LIP method produced very good predictions. In particular for longer loops it outperformed other methods.
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