Resolving the fibrotic niche of human liver cirrhosis at single-cell level.

Liver cirrhosis is a major cause of death worldwide and is characterized by extensive fibrosis. There are currently no effective antifibrotic therapies available. To obtain a better understanding of the cellular and molecular mechanisms involved in disease pathogenesis and enable the discovery of therapeutic targets, here we profile the transcriptomes of more than 100,000 single human cells, yielding molecular definitions for non-parenchymal cell types that are found in healthy and cirrhotic human liver. We identify a scar-associated TREM2+CD9+ subpopulation of macrophages, which expands in liver fibrosis, differentiates from circulating monocytes and is pro-fibrogenic. We also define ACKR1+ and PLVAP+ endothelial cells that expand in cirrhosis, are topographically restricted to the fibrotic niche and enhance the transmigration of leucocytes. Multi-lineage modelling of ligand and receptor interactions between the scar-associated macrophages, endothelial cells and PDGFRα+ collagen-producing mesenchymal cells reveals intra-scar activity of several pro-fibrogenic pathways including TNFRSF12A, PDGFR and NOTCH signalling. Our work dissects unanticipated aspects of the cellular and molecular basis of human organ fibrosis at a single-cell level, and provides a conceptual framework for the discovery of rational therapeutic targets in liver cirrhosis.

A genome-wide association study implicates the APOE locus in nonpathological cognitive ageing.

Cognitive decline is a feared aspect of growing old. It is a major contributor to lower quality of life and loss of independence in old age. We investigated the genetic contribution to individual differences in nonpathological cognitive ageing in five cohorts of older adults. We undertook a genome-wide association analysis using 549 692 single-nucleotide polymorphisms (SNPs) in 3511 unrelated adults in the Cognitive Ageing Genetics in England and Scotland (CAGES) project. These individuals have detailed longitudinal cognitive data from which phenotypes measuring each individual's cognitive changes were constructed. One SNP--rs2075650, located in TOMM40 (translocase of the outer mitochondrial membrane 40 homolog)--had a genome-wide significant association with cognitive ageing (P=2.5 × 10(-8)). This result was replicated in a meta-analysis of three independent Swedish cohorts (P=2.41 × 10(-6)). An Apolipoprotein E (APOE) haplotype (adjacent to TOMM40), previously associated with cognitive ageing, had a significant effect on cognitive ageing in the CAGES sample (P=2.18 × 10(-8); females, P=1.66 × 10(-11); males, P=0.01). Fine SNP mapping of the TOMM40/APOE region identified both APOE (rs429358; P=3.66 × 10(-11)) and TOMM40 (rs11556505; P=2.45 × 10(-8)) as loci that were associated with cognitive ageing. Imputation and conditional analyses in the discovery and replication cohorts strongly suggest that this effect is due to APOE (rs429358). Functional genomic analysis indicated that SNPs in the TOMM40/APOE region have a functional, regulatory non-protein-coding effect. The APOE region is significantly associated with nonpathological cognitive ageing. The identity and mechanism of one or multiple causal variants remain unclear.

More than 1,000 putative new human signalling proteins revealed by EST data mining.

Cloning procedures aided by homology searches of EST databases have accelerated the pace of discovery of new genes, but EST database searching remains an involved and onerous task. More than 1.6 million human EST sequences have been deposited in public databases, making it difficult to identify ESTs that represent new genes. Compounding the problems of scale are difficulties in detection associated with a high sequencing error rate and low sequence similarity between distant homologues. We have developed a new method, coupling BLAST-based searches with a domain identification protocol, that filters candidate homologues. Application of this method in a large-scale analysis of 100 signalling domain families has led to the identification of ESTs representing more than 1,000 novel human signalling genes. The 4,206 publicly available ESTs representing these genes are a valuable resource for rapid cloning of novel human signalling proteins. For example, we were able to identify ESTs of at least 106 new small GTPases, of which 6 are likely to belong to new subfamilies. In some cases, further analyses of genomic DNA led to the discovery of previously unidentified full-length protein sequences. This is exemplified by the in silico cloning (prediction of a gene product sequence using only genomic and EST sequence data) of a new type of GTPase with two catalytic domains.

Cytoplasmic signalling domains: the next generation.

Since the late 1980s, when Src-homology SH2 and SH3 domains were identified, the repertoire of non-catalytic signalling domains has increased to number over 30. As it is expected that further regulatory domains shall be found, unravelling the complex network of their interactions remains an on-going challenge.

JmjC: cupin metalloenzyme-like domains in jumonji, hairless and phospholipase A2beta.

On the basis of significant sequence similarity, we have identified JmjC domains in more than 100 eukaryotic and bacterial sequences. These include human hairless, mutated in individuals with alopecia universalis, retinoblastoma-binding protein 2 and several putative chromatin-associated proteins. JmjC domains are predicted to be metalloenzymes that adopt the cupin fold, and are candidates for enzymes that regulate chromatin remodelling.

Alternative splicing of dystrobrevin regulates the stoichiometry of syntrophin binding to the dystrophin protein complex.

Dystrophin coordinates the assembly of a complex of structural and signalling proteins that is required for normal muscle function. A key component of the dystrophin-associated protein complex (DPC) is alpha-dystrobrevin, a dystrophin-related and -associated protein whose absence results in muscular dystrophy and neuromuscular junction defects [1,2]. The current model of the DPC predicts that dystrophin and dystrobrevin each bind a single syntrophin molecule [3]. The syntrophins are PDZ-domain-containing proteins that facilitate the recruitment of signalling proteins such as nNOS (neuronal nitric oxide synthase) to the DPC [4]. Here we show, using yeast two-hybrid analysis and biochemical binding studies, that alpha-dystrobrevin in fact contains two independent syntrophin-binding sites in tandem. The previously undescribed binding site is situated within an alternatively spliced exon of alpha-dystrobrevin, termed the variable region-3 (vr3) sequence, which is specifically expressed in skeletal and cardiac muscle [5,6]. Analysis of the syntrophin-binding region of dystrobrevin reveals a tandem pair of predicted alpha helices with significant sequence similarity. These alpha helices, each termed a syntrophin-binding motif, are also highly conserved in dystrophin and utrophin. Together these data show that there are four potential syntrophin-binding sites per dystrophin complex in skeletal muscle: two on dystrobrevin and two on dystrophin or utrophin. Furthermore, alternative splicing of dystrobrevin provides a mechanism for regulating the stoichiometry of syntrophin association with the DPC. This is likely to have important consequences for the recruitment of specific signalling molecules to the DPC and ultimately for its function.

Phospholipase D regulation and localisation is dependent upon a phosphatidylinositol 4,5-biphosphate-specific PH domain.

The signalling pathway leading, for example, to actin cytoskeletal reorganisation, secretion or superoxide generation involves phospholipase D (PLD)-catalysed hydrolysis of phosphatidylcholine to generate phosphatidic acid, which appears to mediate the messenger functions of this pathway. Two PLD genes (PLD1 and PLD2) with similar domain structures have been doned and progress has been made in identifying the protein regulators of PLD1 activation, for example Arf and Rho family members. The activities of both PLD isoforms are dependent on phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2) and our sequence analysis suggested the presence of a pleckstrin homology (PH) domain in PLD1, although its absence has also been daimed. Investigation of the inositide dependence showed that a bis-phosphorylated lipid with a vicinal pair of phosphates was required for PLD1 activity. Furthermore, PLD1 bound specifically and with high affinity to lipid surfaces containing PI(4,5)P2 independently of the substrate phosphatidylcholine, suggesting a key role for the PH domain in PLD function. Importantly, a glutathione-S-transferase (GST) fusion protein comprising GST and the PH domain of PLD1 (GST-PLD1-PH) also bound specifically to supported lipid monolayers containing PI(4,5)P2. Point mutations within the PLD1 PH domain inhibited enzyme activity, whereas deletion of the domain both inhibited enzyme activity and disrupted normal PLD1 localisation. Thus, the functional PH domain regulates PLD by mediating its interaction with polyphosphoinositide-containing membranes; this might also induce a conformational change, thereby regulating catalytic activity.

Plagiarized bacterial genes in the human book of life.

The initial analysis of the human genome draft sequence reveals that our 'book of life' is multi-authored. A small but significant proportion of our genes owes their heritage not to antecedent eukaryotes but instead to bacteria. The publicly funded Human Genome Project study indicates that about 0.5% of all human genes were copied into the genome from bacterial sources. Detailed sequence analyses point to these 'horizontal gene transfer' events having occurred relatively recently. So how did the human 'book of life' evolve to be a chimaera, part animal and part bacterium? And what was the probable evolutionary impact of such gene plagiarism?

Protein fold irregularities that hinder sequence analysis.

The detection of homologous protein sequences frequently provides useful predictions of function and structure. Methods for homology searching have continued to improve, such that very distant evolutionary relationships can now be detected. Little attention has been paid, however, to the problems of detecting homology when domains are inserted or permuted. Here we review recent occurrences of these phenomena and discuss methods that permit their detection.

Protein families in multicellular organisms.

The complete sequence of the nematode worm Caenorhabditis elegans contains the genetic machinery that is required to undertake the core biological processes of single cells. However, the genome also encodes proteins that are associated with multicellularity, as well as others that are lineage-specific expansions of phylogenetically widespread families and yet more that are absent in non-nematodes. Ongoing analysis is beginning to illuminate the similarities and differences among human proteins and proteins that are encoded by the genomes of the multicellular worm and the unicellular yeast, and will be essential in determining the reliability of transferring experimental data among phylogenetically distant species.

ELIXIR-UK role in bioinformatics training at the national level and across ELIXIR.

ELIXIR-UK is the UK node of ELIXIR, the European infrastructure for life science data. Since its foundation in 2014, ELIXIR-UK has played a leading role in training both within the UK and in the ELIXIR Training Platform, which coordinates and delivers training across all ELIXIR members. ELIXIR-UK contributes to the Training Platform's coordination and supports the development of training to address key skill gaps amongst UK scientists. As part of this work it acts as a conduit for nationally-important bioinformatics training resources to promote their activities to the ELIXIR community. ELIXIR-UK also leads ELIXIR's flagship Training Portal, TeSS, which collects information about a diverse range of training and makes it easily accessible to the community. ELIXIR-UK also works with others to provide key digital skills training, partnering with the Software Sustainability Institute to provide Software Carpentry training to the ELIXIR community and to establish the Data Carpentry initiative, and taking a lead role amongst national stakeholders to deliver the StaTS project - a coordinated effort to drive engagement with training in statistics.

The crystal structure of the catalytic domain of human urokinase-type plasminogen activator.

BACKGROUND: Urokinase-type plasminogen activator (u-PA) promotes fibrinolysis by catalyzing the conversion of plasminogen to the active protease plasmin via the cleavage of a peptide bond. When localized to the external cell surface it contributes to tissue remodelling and cellular migration; inhibition of its activity impedes the spread of cancer. u-PA has three domains: an N-terminal receptor-binding growth factor domain, a central kringle domain and a C-terminal catalytic protease domain. The biological roles of the fibrinolytic enzymes render them therapeutic targets, however, until now no structure of the protease domain has been available. Solution of the structure of the u-PA serine protease was undertaken to provide such data. RESULTS: The crystal structure of the catalytic domain of recombinant, non-glycosylated human u-PA, complexed with the inhibitor Glu-Gly-Arg chloromethyl ketone (EGRcmk), has been determined at a nominal resolution of 2.5 A and refined to a crystallographic R-factor of 22.4% on all data (20.4% on data > 3 sigma). The enzyme has the expected topology of a trypsin-like serine protease. CONCLUSIONS: The enzyme has an S1 specificity pocket similar to that of trypsin, a restricted, less accessible, hydrophobic S2 pocket and a solvent-accessible S3 pocket which is capable of accommodating a wide range of residues. The EGRcmk inhibitor binds covalently at the active site to form a tetrahedral hemiketal structure. Although the overall structure is similar to that of homologous serine proteases, at six positions insertions of extra residues in loop regions create unique surface areas. One of these loop regions is highly mobile despite being anchored by the disulphide bridge which is characteristic of a small subset of serine proteases namely tissuetype plasminogen activator, Factor XII and Complement Factor I.

Porcine natural-killer-enhancing factor-B: oligomerisation and identification as a calpain substrate in vitro.

Natural-killer-enhancing factor-B (NKEF-B) (monomeric mass = 21.82 kDa) was purified from the cytosol of porcine red blood cells and its identity was established by microsequencing. NKEF-B oligomerisation was investigated by gel filtration and small-angle X-ray scattering (SAXS). Native NKEF-B readily forms disulphide-linked dimers, but when fully reduced, the protein forms discrete oligomers containing 16 +/- 1 monomers. A total of 40% of the purified enzyme was deduced to be cysteinylated, which is consistent with the modification of one or both of two putative active site cysteine residues. In vitro, NKEF-B was found to be a specific substrate of mu- and m-calpains, the calcium-dependent cysteine proteases. The cleavage events were followed by SDS-PAGE and the cleavage sites pinpointed by N-terminally sequencing the resulting digestion fragments. This in vitro cleavage data provides support to the hypothesis that calpromotin (NKEF-B), an erythron peroxiredoxin involved in the regulation of calcium-dependent potassium transport across the plasma membrane, is cleaved by calpain in vivo.

Conformational stability studies of the pleckstrin DEP domain: definition of the domain boundaries.

Pleckstrin is the major substrate of protein kinase C in platelets. It contains at its N- and C-termini two pleckstrin homology (PH) domains which have been proposed to mediate protein-protein and protein-lipid interactions. A new module, called DEP, has recently been identified by sequence analysis in the central region of pleckstrin. In order to study this module, several recombinant polypeptides corresponding to the DEP module and N- and C-termini extended forms have been expressed. Using circular dichroism (CD) and nuclear magnetic resonance (NMR) techniques, the domain boundaries have been determined that yield a soluble and folded pleckstrin DEP domain. This comprises 93 amino acids with an alpha/beta fold in agreement with secondary structure predictions. Stability studies indicate that the regions surrounding the DEP domain do not contribute to its stability suggesting that the phosphorylation sites at S113, T114 and S117 are in an unstructured region. Identification of the regions of pleckstrin that are folded shall facilitate determination of its structure and function.

The compact domain conformation of human Glu-plasminogen in solution.

A complete understanding of the accelerating mechanisms of plasminogen activation and fibrinolysis necessarily requires structural information on the conformational forms of plasminogen. Given the absence of high-resolution structural data on plasminogen the use of lower resolution approaches has been adopted. Two such approaches have previously indicated a compact conformation of Glu-plasminogen (Tranqui, L., Prandini, M., and Chapel, A. (1979) Biol. Cellulaire, 34, 39-42; Bányai, L. and Patthy, L. (1985) Biochim. Biophys. Acta, 832, 224-227) whereas a third has suggested a fairly extended conformation (Mangel, W., Lin, B. and Ramakrishnan, V. (1990) Science, 248, 69-73). Native Glu-plasminogen has been investigated using small-angle X-ray scattering (SAXS) experiments. It is concluded that this molecule in solution is compact (radius of gyration, RG 3.05 +/- 0.02 nm and maximum intramolecular distance, Im 9.1 +/- 0.3 nm) and that the data are consistent with the right-handed spiral structure observed using electron microscopy by Tranqui et al. (1979). A spiral structure of native plasminogen would have important implications for the conformational response of plasminogen to fibrin and concomitant stimulation of plasminogen activation.

Stromal cell-derived receptor 2 and cytochrome b561 are functional ferric reductases.

Iron has a variety of functions in cellular organisms ranging from electron transport and DNA synthesis to adenosine triphosphate (ATP) and neurotransmitter synthesis. Failure to regulate the homeostasis of iron can lead to cognition and demyelination disorders when iron levels are deficient, and to neurodegenerative disorders when iron is in excess. In this study we show that three members of the b561 family of predicted ferric reductases, namely mouse cytochrome b561 and mouse and fly stromal cell-derived receptor 2 (SDR2), have ferric reductase activity. Given that a fourth member, duodenal cytochrome b (Dcytb), has previously been shown to be a ferric reductase, it is likely that all remaining members of this family also exhibit this activity. Furthermore, we show that the rat sdr2 message is predominantly expressed in the liver and kidney, with low expression in the duodenum. In hypotransferrinaemic (hpx) mice, sdr2 expression in the liver and kidney is reduced, suggesting that it may be regulated by iron. Moreover, we demonstrate the presence of mouse sdr2 in the choroid plexus and in the ependymal cells lining the four ventricles, through in situ hybridization analysis.

Molecular characterisation of a thermoactive beta-1,3-glucanase from Oerskovia xanthineolytica.

Molecular characterisation of a lytic thermoactive beta-1,3-glucanase from Oerskovia xanthineolytica LL-G109 has been performed. A molecular mass of 27 195.6 +/- 1.3 Da and an isoelectric point of 4.85 were determined by electrospray mass spectrometry and from its titration curve, respectively. Its thermoactivity profile shows it to be a heat-stable enzyme with a temperature optimum of 65 degrees C. The secondary structure content of the protein was estimated by circular dichroism to be approx. 25% alpha-helix, 7% random coil, and 68% beta-sheet and beta-turn structure. Nuclear magnetic resonance spectra confirm the high content of beta-structure. Furthermore, the presence of a compact hydrophobic core is indicated by the presence of slowly exchanging amide hydrogens and the enzyme's relatively high resistance to proteolysis. The N-terminal sequences of the intact protein and of a tryptic peptide each exhibit significant similarity to family 16 of glycosyl hydrolases whose overall fold is known to contain almost exclusively beta-sheets and surface loops. Moreover, the sequenced tryptic peptide appears to encompass residues of the Oerskovia xanthineolytica glucanase active site, since it contains a portion of the family 16 active-site motif E-[L/I/V]-D-[L/I/V]-E.

Identification of distant homologues of fibroblast growth factors suggests a common ancestor for all beta-trefoil proteins.

Determination of the structures of fibroblast growth factors and interleukin-1s has previously revealed that they both adopt a beta-trefoil fold, similar to those found in Kunitz soybean trypsin inhibitors, ricin-like toxins, plant agglutinins and hisactophilin. These families possess distinct functions and occur in different subcellular localisations, and they appear to lack significant similarities in their sequences, ligands and modes of ligand binding. We have analysed the significance of sequence identities observed after structure alignment and provide statistical evidence that these beta-trefoil proteins are all homologues, having arisen from a common ancestor. In addition, we have explored the sequence space of all beta-trefoil proteins and have determined that the actin-binding proteins fascins, and other proteins of unknown function, are beta-trefoil family homologues. Unlike other beta-trefoil proteins, the triplicated repeats in each of the four beta-trefoil domains of fascins are significantly similar in sequence. This hints at how the beta-trefoil fold arose from the duplication of an ancestral gene encoding a homotrimeric single-repeat protein. The combined analysis of structure and sequence databases for detecting significant similarities is suggested as a highly sensitive approach to determining the common ancestry of extremely divergent homologues.