No abstract

Post-translational modifications (PTMs) occur on almost all proteins analyzed to date. The function of a modified protein is often strongly affected by these modifications and therefore increased knowledge about the potential PTMs of a target protein may increase our understanding of the molecular processes in which it takes part. High-throughput methods for the identification of PTMs are being developed, in particular within the fields of proteomics and mass spectrometry. However, these methods are still in their early stages, and it is indeed advantageous to cut down on the number of experimental steps by integrating computational approaches into the validation procedures. Many advanced methods for the prediction of PTMs exist and many are made publicly available. We describe our experiences with the development of prediction methods for phosphorylation and glycosylation sites and the development of PTM-specific databases. In addition, we discuss novel ideas for PTM visualization (exemplified by kinase landscapes) and improvements for prediction specificity (by using ESS--evolutionary stable sites). As an example, we present a new method for kinase-specific prediction of phosphorylation sites, NetPhosK, which extends our earlier and more general tool, NetPhos. The new server, NetPhosK, is made publicly available at the URL http://www.cbs.dtu.dk/services/NetPhosK/. The issues of underestimation, over-prediction and strategies for improving prediction specificity are also discussed.

We present a sequence-based method, SecretomeP, for the prediction of mammalian secretory proteins targeted to the non-classical secretory pathway, i.e. proteins without an N-terminal signal peptide. So far only a limited number of proteins have been shown experimentally to enter the non-classical secretory pathway. These are mainly fibroblast growth factors, interleukins and galectins found in the extracellular matrix. We have discovered that certain pathway-independent features are shared among secreted proteins. The method presented here is also capable of predicting (signal peptide-containing) secretory proteins where only the mature part of the protein has been annotated or cases where the signal peptide remains uncleaved. By scanning the entire human proteome we identified new proteins potentially undergoing non-classical secretion. Predictions can be made at http://www.cbs.dtu.dk/services/SecretomeP.

Most current approaches for analyzing metagenomic data rely on comparisons to reference genomes, but the microbial diversity of many environments extends far beyond what is covered by reference databases. De novo segregation of complex metagenomic data into specific biological entities, such as particular bacterial strains or viruses, remains a largely unsolved problem. Here we present a method, based on binning co-abundant genes across a series of metagenomic samples, that enables comprehensive discovery of new microbial organisms, viruses and co-inherited genetic entities and aids assembly of microbial genomes without the need for reference sequences. We demonstrate the method on data from 396 human gut microbiome samples and identify 7,381 co-abundance gene groups (CAGs), including 741 metagenomic species (MGS). We use these to assemble 238 high-quality microbial genomes and identify affiliations between MGS and hundreds of viruses or genetic entities. Our method provides the means for comprehensive profiling of the diversity within complex metagenomic samples.

Systematic and quantitative analysis of protein phosphorylation is revealing dynamic regulatory networks underlying cellular responses to environmental cues. However, matching these sites to the kinases that phosphorylate them and the phosphorylation-dependent binding domains that may subsequently bind to them remains a challenge. NetPhorest is an atlas of consensus sequence motifs that covers 179 kinases and 104 phosphorylation-dependent binding domains [Src homology 2 (SH2), phosphotyrosine binding (PTB), BRCA1 C-terminal (BRCT), WW, and 14–3–3]. The atlas reveals new aspects of signaling systems, including the observation that tyrosine kinases mutated in cancer have lower specificity than their non-oncogenic relatives. The resource is maintained by an automated pipe line, which uses phylogenetic trees to structure the currently available in vivo and in vitro data to derive probabilistic sequence models of linear motifs. The atlas is available as a community resource (http://netphorest.info).

Many secretory proteins and peptides are synthesized as inactive precursors that in addition to signal peptide cleavage undergo post-translational processing to become biologically active polypeptides. Precursors are usually cleaved at sites composed of single or paired basic amino acid residues by members of the subtilisin/kexin-like proprotein convertase (PC) family. In mammals, seven members have been identified, with furin being the one first discovered and best characterized. Recently, the involvement of furin in diseases ranging from Alzheimer's disease and cancer to anthrax and Ebola fever has created additional focus on proprotein processing. We have developed a method for prediction of cleavage sites for PCs based on artificial neural networks. Two different types of neural networks have been constructed: a furin-specific network based on experimental results derived from the literature, and a general PC-specific network trained on data from the Swiss-Prot protein database. The method predicts cleavage sites in independent sequences with a sensitivity of 95% for the furin neural network and 62% for the general PC network. The ProP method is made publicly available at http://www.cbs.dtu.dk/services/ProP.

Vultures are scavengers that fill a key ecosystem niche, in which they have evolved a remarkable tolerance to bacterial toxins in decaying meat. Here we report the first deep metagenomic analysis of the vulture microbiome. Through face and gut comparisons of 50 vultures representing two species, we demonstrate a remarkably conserved low diversity of gut microbial flora. The gut samples contained an average of 76 operational taxonomic units (OTUs) per specimen, compared with 528 OTUs on the facial skin. Clostridia and Fusobacteria, widely pathogenic to other vertebrates, dominate the vulture's gut microbiota. We reveal a likely faecal-oral-gut route for their origin. DNA of prey species detectable on facial swabs was completely degraded in the gut samples from most vultures, suggesting that the gastrointestinal tracts of vultures are extremely selective. Our findings show a strong adaption of vultures and their bacteria to their food source, exemplifying a specialized host-microbial alliance.

Picornaviral proteinases are responsible for maturation cleavages of the viral polyprotein, but also catalyze the degradation of cellular targets. Using graphical visualization techniques and neural network algorithms, we have investigated the sequence specificity of the two proteinases 2AP" and 3CPm. The cleavage of VPO (giving rise to VP2 and VP4). which is carried out by a so-far unknown proteinase, was also examined. In combination with a novel surface exposure prediction algorithm, our neural network approach successfully distinguishes known cleavage sites from noncleavage sites and yields a more consistent definition of features common to these sites. The method is able to predict experimentally determined cleavage sites in cellular proteins. We present a list of mammalian and other proteins that are predicted to be possible targets for the viral proteinases. Whether these proteins are indeed cleaved awaits experimental verification. Additionally, we report several errors detected in the protein databases.A computer server for prediction of cleavage sites by picornaviral proteinases is publicly available at the e-mail address NetPicoRNA@cbs.dtu.dk or via WWW at http://www.cbs.dtu.dkfservices/NetPicoRNA.Keywords: cleavage site prediction; neural networks; picornavirus; proteinase; surface exposureMembers of the picornavirus family express their genomic RNA as a single polyprotein that is proteolytically processed to the mature polypeptides. At least three proteinases are required for the individual protein components to be released (reviewed in Krausslich Hellen et al., 1989;Lawson & Semler, 1990). The primary cleavage, which severs the capsid precursor PI from the nonstructural region P2-P3, is performed cotranslationally by the viral proteinase 2APm in enteroviruses and human rhinoviruses (HRVs; see Fig. I). Most of the remaining cleavages are catalyzed by the viral proteinase, 3CP". In cardio-, hepato-, and aphthoviruses, which also belong to the picornavirus family, the L-proteinase performs functions similar to those of 2APm, resulting in a somewhat different cleavage scheme (see Fig. I). Concomitantly with RNA encapsidation, VPO is cleaved to VP4 and VP2; it is believed that the RNA itself exerts a catalytic function in this event (Arnold et a!., 1987;Harber et al., 1991;Bishop &Anderson, 1993;Basavappa et al., 1994).In addition to processing of the viral polyprotein, the proteinases also cleave cellular targets. When infected with poliovirus, at least nine acidic and five basic cellular proteins were shown to be degraded in two-dimensional gel electrophoresis (Urzanqui & Carrasco, 1989). The degradation of cellular proteins seems to be part of the viral attack mechanism, leading to host cell shur-ofJ-a decrease in cellular transcription and translation that has no influence on viral replication. The best-studied event is the cleavage of the eukaryotic initiation factor 4G (eIF-4G). which is required for cap-dependent translation of cellular mRNA. This protein is degraded by 2APm in entero-and...