Kenji Mizuguchi

FlyMine: an integrated database for Drosophila and Anopheles genomics

FlyMine is a data warehouse that addresses one of the important challenges of modern biology: how to integrate and make use of the diversity and volume of current biological data. Its main focus is genomic and proteomics data for Drosophila and other insects. It provides web access to integrated data at a number of different levels, from simple browsing to construction of complex queries, which can be executed on either single items or lists.

Identification and classification of bacterial Type III toxin–antitoxin systems encoded in chromosomal and plasmid genomes

Toxin–antitoxin systems are widespread in bacteria and archaea. They perform diverse functional roles, including the generation of persistence, maintenance of genetic loci and resistance to bacteriophages through abortive infection. Toxin–antitoxin systems have been divided into three types, depending on the nature of the interacting macromolecules. The recently discovered Type III toxin–antitoxin systems encode protein toxins that are inhibited by pseudoknots of antitoxic RNA, encoded by short tandem repeats upstream of the toxin gene. Recent studies have identified the range of Type I and Type II systems within current sequence databases. Here, structure-based homology searches were combined with iterative protein sequence comparisons to obtain a current picture of the prevalence of Type III systems. Three independent Type III families were identified, according to toxin sequence similarity. The three families were found to be far more abundant and widespread than previously known, with examples throughout the Firmicutes, Fusobacteria and Proteobacteria. Functional assays confirmed that representatives from all three families act as toxin–antitoxin loci within Escherichia coli and at least two of the families confer resistance to bacteriophages. This study shows that active Type III toxin–antitoxin systems are far more diverse than previously known, and suggests that more remain to be identified.

Organic Anion Transporting Polypeptides of the OATP/SLCO Superfamily: Identification of New Members in Nonmammalian Species, Comparative Modeling and a Potential Transport Mode

Organic anion-transporting polypeptides (human, OATPs; other animals, Oatps; gene symbol, SLCO/Slco) form a transport protein superfamily that mediates the translocation of amphipathic substrates across the plasma membrane of animal cells. So far, OATPs/Oatps have been identified in human, rat and mouse tissues. In this study, we used bioinformatic tools to detect new members of the OATP/SLCO superfamily in nonmammalian species and to build models for the three-dimensional structure of OATPs/Oatps. New OATP/SLCO superfamily members, some of which form distinct novel families, were identified in chicken, zebrafish, frog, fruit fly and worm species. The lack of OATP/SLCO superfamily members in plants, yeast and bacteria suggests the emergence of an ancient Oatp protein in an early ancestor of the animal kingdom. Structural models were generated for the representative members OATP1B3 and OATP2B1 based on the known structures of the major facilitator superfamily of transport proteins. A model was also built for the large extracellular region between transmembrane helices 9 and 10, following the identification of a novel homology with the Kazal-type serine protease inhibitors. Along with the electrostatic potential and the conservation of key amino acid residues, we propose a common transport mechanism for all OATPs/Oatps, whereby substrates are translocated through a central, positively charged pore in a rocker-switch type of mechanism. Several amino acid residues were identified that may play crucial roles in the proposed transport mechanism.

Getting knotted : a model for the structure and activation of Spatzle

Sequence analyses show that Spätzle, the Drosophila melanogaster Toll-receptor ligand, shows striking similarity to nerve growth factor and coagulogen. Comparative modelling suggests that Spätzle adopts a cystine-knot fold and forms a dimer that contains a single, intermolecular disulphide bridge. Proteolytically cleaved Spätzle could therefore dimerize and activate the Toll receptor by inducing receptor dimerization.

A six-stranded double-psi β barrel is shared by several protein superfamilies

Abstract Background: Six-stranded β barrels with a pseudo-twofold axis are found in several proteins. One group comprises a Greek-key structure with all strands antiparallel; an example is the N-terminal domain of ferredoxin reductase. Others involve parallel strands forming two psi structures (the double-psi β barrel). A recently discovered example of the latter class is aspartate-α-decarboxylase (ADC) from Escherichia coli , a pyruvoyl-dependent tetrameric enzyme involved in the synthesis of pantothenate. Results: Visual inspection and automated database searches identified the six-stranded double-psi β barrel in ADC, Rhodobacter sphaeroides dimethylsulfoxide (DMSO) reductase, E. coli formate dehydrogenase H (FDH H ), the plant defense protein barwin, Humicola insolens endoglucanase V (EGV) and, with a circular permutation, in the aspartic proteinases. Structure-based sequence alignments revealed several interactions including hydrophobic contacts or sidechain–mainchain hydrogen bonds that position the middle β strand under a psi loop, which may significantly contribute to stabilizing the fold. The identification of key interactions allowed the filtering of weak sequence similarities to some of these proteins, which had been detected by sequence database searches. This led to the prediction of the double-psi β-barrel domain in several families of proteins in eukaryotes and archaea. Conclusions: The structure comparison and clustering study of double-psi β barrels suggests that there could be a common homodimeric ancestor to ADC, FDH H and DMSO reductase, and also to barwin and EGV. There are other protein families with unknown structure that are likely to adopt the same fold. In the known structures, the protein active sites cluster around the psi loop, indicating that its rigidity, protrusion and free mainchain functional groups may be well suited to providing a framework for catalysis.

HOMSTRAD: recent developments of the Homologous Protein Structure Alignment Database

HOMSTRAD (http://www-cryst.bioc.cam.ac.uk/ homstrad/) is a collection of protein families, clustered on the basis of sequence and structural similarity. The database is unique in that the protein family sequence alignments have been specially annotated using the program, JOY, to highlight a wide range of structural features. Such data are useful for identifying key structurally conserved residues within the families. Superpositions of the structures within each family are also available and a sensitive structure-aided search engine, FUGUE, can be used to search the database for matches to a query protein sequence. Historically, HOMSTRAD families were generated using several key pieces of software, including COMPARER and MNYFIT, and held in a number of flat files and indexes. A new relational database version of HOMSTRAD, HOMSTRAD BETA (http://www-cryst.bioc.cam. ac.uk/homstradbeta/) is being developed using MySQL. This relational data structure provides more flexibility for future developments, reduces update times and makes data more easily accessible. Consequently it has been possible to add a number of new web features including a custom alignment facility. Altogether, this makes HOMSTRAD and its new BETA version, an excellent resource both for comparative modelling and for identifying distant sequence/structure similarities between proteins.

Drosophila Neurotrophins Reveal a Common Mechanism for Nervous System Formation

Neurotrophic interactions occur in Drosophila, but to date, no neurotrophic factor had been found. Neurotrophins are the main vertebrate secreted signalling molecules that link nervous system structure and function: they regulate neuronal survival, targeting, synaptic plasticity, memory and cognition. We have identified a neurotrophic factor in flies, Drosophila Neurotrophin (DNT1), structurally related to all known neurotrophins and highly conserved in insects. By investigating with genetics the consequences of removing DNT1 or adding it in excess, we show that DNT1 maintains neuronal survival, as more neurons die in DNT1 mutants and expression of DNT1 rescues naturally occurring cell death, and it enables targeting by motor neurons. We show that Spätzle and a further fly neurotrophin superfamily member, DNT2, also have neurotrophic functions in flies. Our findings imply that most likely a neurotrophin was present in the common ancestor of all bilateral organisms, giving rise to invertebrate and vertebrate neurotrophins through gene or whole-genome duplications. This work provides a missing link between aspects of neuronal function in flies and vertebrates, and it opens the opportunity to use Drosophila to investigate further aspects of neurotrophin function and to model related diseases.

A model of a transmembrane drug-efflux pump from Gram-negative bacteria.

In Gram‐negative bacteria, drug resistance is due in part to the activity of transmembrane efflux‐pumps, which are composed of three types of proteins. A representative pump from Escherichia coli is an assembly of the trimeric outer‐membrane protein TolC, which is an allosteric channel, the trimeric inner‐membrane proton‐antiporter AcrB, and the periplasmic protein, AcrA. The pump displaces drugs vectorially from the bacterium using proton electrochemical force. Crystal structures are available for TolC and AcrB from E. coli, and for the AcrA homologue MexA from Pseudomonas aeruginosa. Based on homology modelling and molecular docking, we show how AcrA, AcrB and TolC might assemble to form a tripartite pump, and how allostery may occur during transport.

CAMPASS: a database of structurally aligned protein superfamilies

We thank Andrej Sali, John Overington and Mark Johnson for use of the programs COMPARER, JOY and MALIGN, respectively. We thank the anonymous referees for their useful comments. For financial support, RS thanks the Imperial Cancer Research Fund; DFB and HAN Oxford Molecular Ltd; KM HFSP; NS The Wellcome Trust; and RES the MRC.

A family of proteins related to Spätzle, the toll receptor ligand, are encoded in the Drosophila genome.

The Drosophila gene Spätzle encodes the activating ligand for the Toll receptor. This signaling pathway is required for dorso‐ventral patterning in the early embryo and an antifungal immune response in larvae and adults. The genome sequence of Drosophila shows that there are a total of eight Toll‐like receptors and these may function in other aspects of embryonic development and innate immunity. Here we describe five Drosophila homologues of Spätzle (Spz2‐6) found using an iterative searching method. All five appear to encode proteins containing neurotrophin‐like cystine‐knot domains. In addition, most retain a characteristic intron‐exon structure shared with the prototype Spätzle gene. This provides evidence that the family arose by ancient gene duplication events and indicates that the gene products may represent activating ligands for corresponding Toll receptors. Expression studies show that only Spz4 is expressed strongly in larvae and adults and thus may be involved in an ancillary antifungal response mediated by Toll‐5. By contrast, Spz6 shows a complex spatial and temporally regulated expression pattern in the late embryo. Thus the new Toll/Spätzle families of signaling molecules may have important roles in other aspects of development and immunity. Proteins 2001;45:71–80.