Protein features as determinants of wild-type glycoside hydrolase thermostability.

Thermostable enzymes for conversion of lignocellulosic biomass into biofuels have significant advantages over enzymes with more moderate themostability due to the challenging application conditions. Experimental discovery of thermostable enzymes is highly cost intensive, and the development of in-silico methods guiding the discovery process would be of high value. To develop such an in-silico method and provide the data foundation of it, we determined the melting temperatures of 602 fungal glycoside hydrolases from the families GH5, 6, 7, 10, 11, 43, and AA9 (formerly GH61). We, then used sequence and homology modeled structure information of these enzymes to develop the ThermoP melting temperature prediction method. Futhermore, in the context of thermostability, we determined the relative importance of 160 molecular features, such as amino acid frequencies and spatial interactions, and exemplified their biological significance. The presented prediction method is made publicly available at http://www.cbs.dtu.dk/services/ThermoP.

Cofactory: sequence-based prediction of cofactor specificity of Rossmann folds.

Obtaining optimal cofactor balance to drive production is a challenge in metabolically engineered microbial production strains. To facilitate identification of heterologous enzymes with desirable altered cofactor requirements from native content, we have developed Cofactory, a method for prediction of enzyme cofactor specificity using only primary amino acid sequence information. The algorithm identifies potential cofactor binding Rossmann folds and predicts the specificity for the cofactors FAD(H2), NAD(H), and NADP(H). The Rossmann fold sequence search is carried out using hidden Markov models whereas artificial neural networks are used for specificity prediction. Training was carried out using experimental data from protein-cofactor structure complexes. The overall performance was benchmarked against an independent evaluation set obtaining Matthews correlation coefficients of 0.94, 0.79, and 0.65 for FAD(H2), NAD(H), and NADP(H), respectively. The Cofactory method is made publicly available at http://www.cbs.dtu.dk/services/Cofactory.

Bacterial diversity in snow on North Pole ice floes.

The microbial abundance and diversity in snow on ice floes at three sites near the North Pole was assessed using quantitative PCR and 454 pyrosequencing. Abundance of 16S rRNA genes in the samples ranged between 43 and 248 gene copies per millilitre of melted snow. A total of 291,331 sequences were obtained through 454 pyrosequencing of 16S rRNA genes, resulting in 984 OTUs at 97 % identity. Two sites were dominated by Cyanobacteria (72 and 61 %, respectively), including chloroplasts. The third site differed by consisting of 95 % Proteobacteria. Principal component analysis showed that the three sites clustered together when compared to the underlying environments of sea ice and ocean water. The Shannon indices ranged from 2.226 to 3.758, and the Chao1 indices showed species richness between 293 and 353 for the three samples. The relatively low abundances and diversity found in the samples indicate a lower rate of microbial input to this snow habitat compared to snow in the proximity of terrestrial and anthropogenic sources of microorganisms. The differences in species composition and diversity between the sites show that apparently similar snow habitats contain a large variation in biodiversity, although the differences were smaller than the differences to the underlying environment. The results support the idea that a globally distributed community exists in snow and that the global snow community can in part be attributed to microbial input from the atmosphere.

Wide distribution of closely related, antibiotic-producing Arthrobacter strains throughout the Arctic Ocean.

We isolated 16 antibiotic-producing bacterial strains throughout the central Arctic Ocean, including seven Arthrobacter spp. with almost identical 16S rRNA gene sequences. These strains were numerically rare, as revealed using 454 pyrosequencing libraries. Arthrobacter spp. produced arthrobacilins A to C under different culture conditions, but other, unidentified compounds likely contributed to their antibiotic activity.

Predicted coronavirus Nsp5 protease cleavage sites in the human proteome.

BACKGROUND: The coronavirus nonstructural protein 5 (Nsp5) is a cysteine protease required for processing the viral polyprotein and is therefore crucial for viral replication. Nsp5 from several coronaviruses have also been found to cleave host proteins, disrupting molecular pathways involved in innate immunity. Nsp5 from the recently emerged SARS-CoV-2 virus interacts with and can cleave human proteins, which may be relevant to the pathogenesis of COVID-19. Based on the continuing global pandemic, and emerging understanding of coronavirus Nsp5-human protein interactions, we set out to predict what human proteins are cleaved by the coronavirus Nsp5 protease using a bioinformatics approach. RESULTS: Using a previously developed neural network trained on coronavirus Nsp5 cleavage sites (NetCorona), we made predictions of Nsp5 cleavage sites in all human proteins. Structures of human proteins in the Protein Data Bank containing a predicted Nsp5 cleavage site were then examined, generating a list of 92 human proteins with a highly predicted and accessible cleavage site. Of those, 48 are expected to be found in the same cellular compartment as Nsp5. Analysis of this targeted list of proteins revealed molecular pathways susceptible to Nsp5 cleavage and therefore relevant to coronavirus infection, including pathways involved in mRNA processing, cytokine response, cytoskeleton organization, and apoptosis. CONCLUSIONS: This study combines predictions of Nsp5 cleavage sites in human proteins with protein structure information and protein network analysis. We predicted cleavage sites in proteins recently shown to be cleaved in vitro by SARS-CoV-2 Nsp5, and we discuss how other potentially cleaved proteins may be relevant to coronavirus mediated immune dysregulation. The data presented here will assist in the design of more targeted experiments, to determine the role of coronavirus Nsp5 cleavage of host proteins, which is relevant to understanding the molecular pathology of coronavirus infection.

NetPhosBac - a predictor for Ser/Thr phosphorylation sites in bacterial proteins.

There is ample evidence for the involvement of protein phosphorylation on serine/threonine/tyrosine in bacterial signaling and regulation, but very few exact phosphorylation sites have been experimentally determined. Recently, gel-free high accuracy MS studies reported over 150 phosphorylation sites in two bacterial model organisms Bacillus subtilis and Escherichia coli. Interestingly, the analysis of these phosphorylation sites revealed that most of them are not characteristic for eukaryotic-type protein kinases, which explains the poor performance of eukaryotic data-trained phosphorylation predictors on bacterial systems. We used these large bacterial datasets and neural network algorithms to create the first bacteria-specific protein phosphorylation predictor: NetPhosBac. With respect to predicting bacterial phosphorylation sites, NetPhosBac significantly outperformed all benchmark predictors. Moreover, NetPhosBac predictions of phosphorylation sites in E. coli proteins were experimentally verified on protein and site-specific levels. In conclusion, NetPhosBac clearly illustrates the advantage of taxa-specific predictors and we hope it will provide a useful asset to the microbiological community.

Kinase-specific prediction of protein phosphorylation sites.

As extensive mass spectrometry-based mapping of the phosphoproteome progresses, computational analysis of phosphorylation-dependent signaling becomes increasingly important. The linear sequence motifs that surround phosphorylated residues have successfully been used to characterize kinase-substrate specificity. Here, we briefly describe the available resources for predicting kinase-specific phosphorylation from sequence properties. We address the strengths and weaknesses of these resources, which are based on methods ranging from simple consensus patterns to more advanced machine-learning algorithms. Furthermore, a protocol for the use of the artificial neural network based predictors, NetPhos and NetPhosK, is provided. Finally, we point to possible developments with the intention of providing the community with improved and additional phosphorylation predictors for large-scale modeling of cellular signaling networks.

NetPhosYeast: prediction of protein phosphorylation sites in yeast.

UNLABELLED: We here present a neural network-based method for the prediction of protein phosphorylation sites in yeast--an important model organism for basic research. Existing protein phosphorylation site predictors are primarily based on mammalian data and show reduced sensitivity on yeast phosphorylation sites compared to those in humans, suggesting the need for an yeast-specific phosphorylation site predictor. NetPhosYeast achieves a correlation coefficient close to 0.75 with a sensitivity of 0.84 and specificity of 0.90 and outperforms existing predictors in the identification of phosphorylation sites in yeast. AVAILABILITY: The NetPhosYeast prediction service is available as a public web server at http://www.cbs.dtu.dk/services/NetPhosYeast/.

Complete mitochondrial genome of the Oriental Hornet, Vespa orientalis F. (Hymenoptera: Vespidae).

The Oriental Hornet (Vespa orientalis) is a social insect belonging to the Vespiade family (Wasps, Hornets, Yellowjackets), genus Vespa (true Hornets). The oriental hornet is a scavenger and an agricultural pest, especially to bee farmers, but is also recently described as a harvester of solar energy. Here, we report the mitochondrial genome sequence of the Oriental Hornet, Vespa orientalis F., which may play a vital role in understanding this wasp biology, light trapping and generation of electricity. The mitochondrial genome of this hornet is 16,099 bp in length, containing 13 protein-coding genes, 21 transfer RNA genes, and 2 ribosomal RNA genes. The overall base composition of the heavy-strand is 40.3% A, 5.9% C, 13.2% G, and 40.6% T, the percentages of A and T being higher than that of G and C. The mitochondrial genome of the Oriental Hornet, Vespa orientalis F. represents the first mitogenome of a solar energy harvesting insect.

Coronavirus 3CLpro proteinase cleavage sites: possible relevance to SARS virus pathology.

BACKGROUND: Despite the passing of more than a year since the first outbreak of Severe Acute Respiratory Syndrome (SARS), efficient counter-measures are still few and many believe that reappearance of SARS, or a similar disease caused by a coronavirus, is not unlikely. For other virus families like the picornaviruses it is known that pathology is related to proteolytic cleavage of host proteins by viral proteinases. Furthermore, several studies indicate that virus proliferation can be arrested using specific proteinase inhibitors supporting the belief that proteinases are indeed important during infection. Prompted by this, we set out to analyse and predict cleavage by the coronavirus main proteinase using computational methods. RESULTS: We retrieved sequence data on seven fully sequenced coronaviruses and identified the main 3CL proteinase cleavage sites in polyproteins using alignments. A neural network was trained to recognise the cleavage sites in the genomes obtaining a sensitivity of 87.0% and a specificity of 99.0%. Several proteins known to be cleaved by other viruses were submitted to prediction as well as proteins suspected relevant in coronavirus pathology. Cleavage sites were predicted in proteins such as the cystic fibrosis transmembrane conductance regulator (CFTR), transcription factors CREB-RP and OCT-1, and components of the ubiquitin pathway. CONCLUSIONS: Our prediction method NetCorona predicts coronavirus cleavage sites with high specificity and several potential cleavage candidates were identified which might be important to elucidate coronavirus pathology. Furthermore, the method might assist in design of proteinase inhibitors for treatment of SARS and possible future diseases caused by coronaviruses. It is made available for public use at our website: http://www.cbs.dtu.dk/services/NetCorona/.

Phospho.ELM: a database of experimentally verified phosphorylation sites in eukaryotic proteins.

BACKGROUND: Post-translational phosphorylation is one of the most common protein modifications. Phosphoserine, threonine and tyrosine residues play critical roles in the regulation of many cellular processes. The fast growing number of research reports on protein phosphorylation points to a general need for an accurate database dedicated to phosphorylation to provide easily retrievable information on phosphoproteins. DESCRIPTION: Phospho.ELM http://phospho.elm.eu.org is a new resource containing experimentally verified phosphorylation sites manually curated from the literature and is developed as part of the ELM (Eukaryotic Linear Motif) resource. Phospho.ELM constitutes the largest searchable collection of phosphorylation sites available to the research community. The Phospho.ELM entries store information about substrate proteins with the exact positions of residues known to be phosphorylated by cellular kinases. Additional annotation includes literature references, subcellular compartment, tissue distribution, and information about the signaling pathways involved as well as links to the molecular interaction database MINT. Phospho.ELM version 2.0 contains 1703 phosphorylation site instances for 556 phosphorylated proteins. CONCLUSION: Phospho.ELM will be a valuable tool both for molecular biologists working on protein phosphorylation sites and for bioinformaticians developing computational predictions on the specificity of phosphorylation reactions.

Prediction of proprotein convertase cleavage sites.

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.

Feature-based prediction of non-classical and leaderless protein secretion.

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.

The microbiome of New World vultures.

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.

Identification and assembly of genomes and genetic elements in complex metagenomic samples without using reference genomes.

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.

The SH2 domain interaction landscape.

Members of the SH2 domain family modulate signal transduction by binding to short peptides containing phosphorylated tyrosines. Each domain displays a distinct preference for the sequence context of the phosphorylated residue. We have developed a high-density peptide chip technology that allows for probing of the affinity of most SH2 domains for a large fraction of the entire complement of tyrosine phosphopeptides in the human proteome. Using this technique, we have experimentally identified thousands of putative SH2-peptide interactions for more than 70 different SH2 domains. By integrating this rich data set with orthogonal context-specific information, we have assembled an SH2-mediated probabilistic interaction network, which we make available as a community resource in the PepspotDB database. A predicted dynamic interaction between the SH2 domains of the tyrosine phosphatase SHP2 and the phosphorylated tyrosine in the extracellular signal-regulated kinase activation loop was validated by experiments in living cells.

Microbial community structure of Arctic multiyear sea ice and surface seawater by 454 sequencing of the 16S RNA gene.

Dramatic decreases in the extent of Arctic multiyear ice (MYI) suggest this environment may disappear as early as 2100, replaced by ecologically different first-year ice. To better understand the implications of this loss on microbial biodiversity, we undertook a detailed census of the microbial community in MYI at two sites near the geographic North Pole using parallel tag sequencing of the 16S rRNA gene. Although the composition of the MYI microbial community has been characterized by previous studies, microbial community structure has not been. Although richness was lower in MYI than in underlying surface water, we found diversity to be comparable using the Simpson and Shannon's indices (for Simpson t=0.65, P=0.56; for Shannon t=0.25, P=0.84 for a Student's t-test of mean values). Cyanobacteria, comprising 6.8% of reads obtained from MYI, were observed for the first time in Arctic sea ice. In addition, several low-abundance clades not previously reported in sea ice were present, including the phylum TM7 and the classes Spartobacteria and Opitutae. Members of Coraliomargarita, a recently described genus of the class Opitutae, were present in sufficient numbers to suggest niche occupation within MYI.

Motif decomposition of the phosphotyrosine proteome reveals a new N-terminal binding motif for SHIP2.

Advances in mass spectrometry-based proteomics have yielded a substantial mapping of the tyrosine phosphoproteome and thus provided an important step toward a systematic analysis of intracellular signaling networks in higher eukaryotes. In this study we decomposed an uncharacterized proteomics data set of 481 unique phosphotyrosine (Tyr(P)) peptides by sequence similarity to known ligands of the Src homology 2 (SH2) and the phosphotyrosine binding (PTB) domains. From 20 clusters we extracted 16 known and four new interaction motifs. Using quantitative mass spectrometry we pulled down Tyr(P)-specific binding partners for peptides corresponding to the extracted motifs. We confirmed numerous previously known interaction motifs and found 15 new interactions mediated by phosphosites not previously known to bind SH2 or PTB. Remarkably, a novel hydrophobic N-terminal motif ((L/V/I)(L/V/I)pY) was identified and validated as a binding motif for the SH2 domain-containing inositol phosphatase SHIP2. Our decomposition of the in vivo Tyr(P) proteome furthermore suggests that two-thirds of the Tyr(P) sites mediate interaction, whereas the remaining third govern processes such as enzyme activation and nucleic acid binding.

Linear motif atlas for phosphorylation-dependent signaling.

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 pipeline, 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).