Publications

Cleavage sites in nuclear-encoded mitochondrial protein targeting peptides (mTPs) from mammals, yeast, and plants have been analysed for characteristic physicochemical features using statistical methods, perceptrons, multilayer neural networks, and self-organizing feature maps. Three different sequence motifs were found, revealing loosely defined arginine motifs with Arg in positions -10, -3, and -2. A self-organizing feature map was able to cluster these three types of endopeptidase target sites but did not identify any species-specific characteristics in mTPs. Neural networks were used to define local sequence features around precursor cleavage sites.

The annotation efforts of the BIOSAPIENS European Network of Excellence have generated several distributed annotation systems (DAS) with the aim of integrating Bioinformatics resources and annotating metazoan genomes ( http://www.biosapiens.info ). In this context, the PONGO DAS server ( http://pongo.biocomp.unibo.it ) provides the annotation on predictive basis for the all-alpha membrane proteins in the human genome, not only through DAS queries, but also directly using a simple web interface. In order to produce a more comprehensive analysis of the sequence at hand, this annotation is carried out with four selected and high scoring predictors: TMHMM2.0, MEMSAT, PRODIV and ENSEMBLE1.0. The stored and pre-computed predictions for the human proteins can be searched and displayed in a graphical view. However the web service allows the prediction of the topology of any kind of putative membrane proteins, regardless of the organism and more importantly with the same sequence profile for a given sequence when required. Here we present a new web server that incorporates the state-of-the-art topology predictors in a single framework, so that putative users can interactively compare and evaluate four predictions simultaneously for a given sequence. Together with the predicted topology, the server also displays a signal peptide prediction determined with SPEP. The PONGO web server is available at http://pongo.biocomp.unibo.it/pongo .

No abstract is provided for this article.

Like all other secretory proteins, the HIV-1 envelope glycoprotein gp160 is targeted to the endoplasmic reticulum (ER) by its signal peptide during synthesis. Proper gp160 folding in the ER requires core glycosylation, disulfide-bond formation and proline isomerization. Signal-peptide cleavage occurs only late after gp160 chain termination and is dependent on folding of the soluble subunit gp120 to a near-native conformation. We here detail the mechanism by which co-translational signal-peptide cleavage is prevented. Conserved residues from the signal peptide and residues downstream of the canonical cleavage site form an extended alpha-helix in the ER membrane, which covers the cleavage site, thus preventing cleavage. A point mutation in the signal peptide breaks the alpha helix allowing co-translational cleavage. We demonstrate that postponed cleavage of gp160 enhances functional folding of the molecule. The change to early cleavage results in decreased viral fitness compared to wild-type HIV.

No abstract is provided for this article.

In bioinformatics, machine learning methods have been used to predict features embedded in the sequences. In contrast to what is generally assumed, machine learning approaches can also provide new insights into the underlying biology. Here, we demonstrate this by presenting TargetP 2.0, a novel state of art method to identify N-terminal sorting signals, which direct proteins to the secretory pathway, mitochondria and chloroplasts or other plastids. By examining the strongest signals from the attention layer in the network, we find that the second residue in the protein, i.e. the one following the initial methionine, has a strong influence on the classification. When subsequently examining all targeting peptides, we observe that two-thirds of chloroplast and thylakoid transit peptides have an alanine in position two, but only 20% of other plant proteins. Further highlighting the importance of the second residue, we also note that in fungi and single-celled eukaryotes, less than 30% of the targeting peptides have an amino acid that allows the removal of the N-terminal methionine compared with 60% for the proteins without targeting peptide. TargetP 2.0 is available at http://www.cbs.dtu.dk/services/TargetP-2.0/index.php

Many secretory and membrane proteins are N-glycosylated by the oligosaccharyl transferase complex during their translocation across the endoplasmic reticulum membrane. Several experimental observations suggest that the highly conserved STT3 subunit contains the active site of the oligosaccharyl transferase. Here, we report a detailed study of the interaction between the active site of the STT3 protein and nascent polypeptide chains using an in vitro photocrosslinking technique. Our results show that the addition of a glycan moiety in a stretch of approximately 15 residues surrounding a QK(*)T cross-linking site impairs the interaction between the nascent chain and STT3.

A new, simple method for predicting transmembrane segments in integral membrane proteins has been developed. It is based on low-stringency dot-plots of the query sequence against a collection of non-homologous membrane proteins using a previously derived scoring matrix [Cserzö et al., 1994, J. Mol. Biol., 243, 388-396]. This so-called dense alignment surface (DAS) method is shown to perform on par with earlier methods that require extra information in the form of multiple sequence alignments or the distribution of positively charged residues outside the transmembrane segments, and thus improves prediction abilities when only single-sequence information is available or for classes of membrane proteins that do not follow the 'positive inside' rule.

A topology map of a membrane protein defines the location of transmembrane helices and the orientation of soluble domains relative to the membrane. In the absence of a high‐resolution structure, a topology map is an essential guide for studying structure–function relationships. Although these maps can be predicted directly from amino acid sequence, the predictions are more accurate if combined with experimental data, which are usually obtained by fusing a reporter protein to the C‐terminus of the protein. However, as reporter proteins are large, they cannot be used to report on the cytoplasmic/periplasmic location of the N‐terminus of a protein. Here, we show that the bimolecular split‐green fluorescent protein complementation system can overcome this limitation and can be used to determine the location of both the N‐ and C‐termini of inner membrane proteins in Escherichia coli .

Most secretion pathways in bacteria and eukaryotic cells are challenged by the requirement for their substrate proteins to mature after they traverse a membrane barrier and enter a reactive oxidizing environment. For Gram-positive bacteria, the mechanisms that protect their exported proteins from misoxidation during their post-translocation maturation are poorly understood. To address this, we separated numerous bacterial species according to their tolerance for oxygen and divided their proteomes based on the predicted subcellular localization of their proteins. We then applied a previously established computational approach that utilizes cysteine incorporation patterns in proteins as an indicator of enzymatic systems that may exist in each species. The Sec-dependent exported proteins from aerobic Gram-positive Actinobacteria were found to encode cysteines in an even-biased pattern indicative of a functional disulfide bond formation system. In contrast, aerobic Gram-positive Firmicutes favor the exclusion of cysteines from both their cytoplasmic proteins and their substantially longer exported proteins. Supporting these findings, we show that Firmicutes, but not Actinobacteria, tolerate growth in reductant. We further demonstrate that the actinobacterium Corynebacterium glutamicum possesses disulfide-bonded proteins and two dimeric Dsb-like enzymes that can efficiently catalyze the formation of disulfide bonds. Our results suggest that cysteine exclusion is an important adaptive strategy against the challenges presented by oxidative environments.