Large-scale benchmark of Endeavour using MetaCore maps.

SUMMARY: Endeavour is a tool that detects the most promising genes within large lists of candidates with respect to a biological process of interest and by combining several genomic data sources. We have benchmarked Endeavour using 450 pathway maps and 826 disease marker sets from MetaCore of GeneGo, Inc. containing a total of 9911 and 12 432 genes, respectively. We obtained an area under the receiver operating characteristic curves of 0.97 for pathway and of 0.91 for disease gene sets. These results indicate that Endeavour can be used to efficiently prioritize candidate genes for pathways and diseases. AVAILABILITY: Endeavour is available at http://www.esat.kuleuven.be/endeavour

A novel microarray approach reveals new tissue-specific signatures of known and predicted mammalian microRNAs.

Microarrays to examine the global expression levels of microRNAs (miRNAs) in a systematic in-parallel manner have become important tools to help unravel the functions of miRNAs and to understand their roles in RNA-based regulation and their implications in human diseases. We have established a novel miRNA-specific microarray platform that enables the simultaneous expression analysis of both known and predicted miRNAs obtained from human or mouse origin. Chemically modified 2'-O-(2-methoxyethyl)-(MOE) oligoribonucleotide probes were arrayed onto Evanescent Resonance (ER) microchips by robotic spotting. Supplementing the complementary probes against miRNAs with carefully designed mismatch controls allowed for accurate sequence-specific determination of miRNA expression profiles obtained from a panel of mouse tissues. This revealed new expression signatures of known miRNAs as well as of novel miRNAs previously predicted using bioinformatic methods. Systematic confirmation of the array data with northern blotting and, in particular, real-time PCR suggests that the described microarray platform is a powerful tool to analyze miRNA expression patterns with rapid throughput and high fidelity.

Signal peptide peptidase dependent cleavage of type II transmembrane substrates releases intracellular and extracellular signals.

The intramembrane-cleaving proteases (I-CLiPs) presenilin-1 and -2 (PS1 and PS2), signal peptide peptidase (SPP) and the Site-2 protease (S2P) catalyze critical steps in cell signaling and are implicated in diseases such as Alzheimer's disease, hepatitis C virus (HCV) infection and cholesterol homeostasis. Here we describe the development of a cellular assay based on cleavage of the transmembrane sequence of the HCV core protein precursor, releasing intra- and extra-cellular signals that represent sequential signal peptidase and SPP cleavage, respectively. We find that the SPP inhibitor (Z-LL)2-ketone (IC50 = 1.33 microM) and the gamma-secretase potent inhibitors NVP-AHW700-NX (IC50 = 51 nM) and LY411575 (IC50 = 61 nM) but not DAPT dose dependently inhibited SPP but not signal peptidase cleavage. Our data confirm that type II orientated substrates, like the HCV transmembrane sequence, are sequentially cleaved by signal peptidase then SPP. This dual assay provides a powerful tool to pharmacologically analyze sequential cleavage events of signal peptidase and SPP and their regulation.

Consensus analysis of signal peptide peptidase and homologous human aspartic proteases reveals opposite topology of catalytic domains compared with presenilins.

The human genome encodes seven intramembrane-cleaving GXGD aspartic proteases. These are the two presenilins that activate signaling molecules and are implicated in Alzheimer's disease, signal peptide peptidase (SPP), required for immune surveillance, and four SPP-like candidate proteases (SPPLs), of unknown function. Here we describe a comparative analysis of the topologies of SPP and its human homologues, SPPL2a, -2b, -2c, and -3. We demonstrate that their N-terminal extensions are located in the extracellular space and, except for SPPL3, are modified with N-glycans. Whereas SPPL2a, -2b, and -2c contain a signal sequence, SPP and SPPL3 contain a type I signal anchor sequence for initiation of protein translocation and membrane insertion. The hydrophilic loops joining the transmembrane regions, which contain the catalytic residues, are facing the exoplasm. The C termini of all these proteins are exposed toward the cytosol. Taken together, our study demonstrates that SPP and its homologues are all of the same principal structure with a catalytic domain embedded in the membrane in opposite orientation to that of presenilins. Other than presenilins, SPPL2a, -2b, -2c, and -3 are therefore predicted to cleave type II-oriented substrate peptides like the prototypic protease SPP.