Range-Wide Sex-Chromosome Sequence Similarity Supports Occasional XY Recombination in European Tree Frogs (Hyla arborea).

In contrast with mammals and birds, most poikilothermic vertebrates feature structurally undifferentiated sex chromosomes, which may result either from frequent turnovers, or from occasional events of XY recombination. The latter mechanism was recently suggested to be responsible for sex-chromosome homomorphy in European tree frogs (Hyla arborea). However, no single case of male recombination has been identified in large-scale laboratory crosses, and populations from NW Europe consistently display sex-specific allelic frequencies with male-diagnostic alleles, suggesting the absence of recombination in their recent history. To address this apparent paradox, we extended the phylogeographic scope of investigations, by analyzing the sequences of three sex-linked markers throughout the whole species distribution. Refugial populations (southern Balkans and Adriatic coast) show a mix of X and Y alleles in haplotypic networks, and no more within-individual pairwise nucleotide differences in males than in females, testifying to recurrent XY recombination. In contrast, populations of NW Europe, which originated from a recent postglacial expansion, show a clear pattern of XY differentiation; the X and Y gametologs of the sex-linked gene Med15 present different alleles, likely fixed by drift on the front wave of expansions, and kept differentiated since. Our results support the view that sex-chromosome homomorphy in H. arborea is maintained by occasional or historical events of recombination; whether the frequency of these events indeed differs between populations remains to be clarified.

The extended GLUT-family of sugar/polyol transport facilitators: nomenclature, sequence characteristics, and potential function of its novel members (review).

During the last 2 years, several novel genes that encode glucose transporter-like proteins have been identified and characterized. Because of their sequence similarity with GLUT1, these genes appear to belong to the family of solute carriers 2A (SLC2A, protein symbol GLUT). Sequence comparisons of all 13 family members allow the definition of characteristic sugar/polyol transporter signatures: (1) the presence of 12 membrane-spanning helices, (2) seven conserved glycine residues in the helices, (3) several basic and acidic residues at the intracellular surface of the proteins, (4) two conserved tryptophan residues, and (5) two conserved tyrosine residues. On the basis of sequence similarities and characteristic elements, the extended GLUT family can be divided into three subfamilies, namely class I (the previously known glucose transporters GLUT1-4), class II (the previously known fructose transporter GLUT5, the GLUT7, GLUT9 and GLUT11), and class III (GLUT6, 8, 10, 12, and the myo-inositol transporter HMIT1). Functional characteristics have been reported for some of the novel GLUTs. Like GLUT1-4, they exhibit a tissue/cell-specific expression (GLUT6, leukocytes, brain; GLUT8, testis, blastocysts, brain, muscle, adipocytes; GLUT9, liver, kidney; GLUT10, liver, pancreas; GLUT11, heart, skeletal muscle). GLUT6 and GLUT8 appear to be regulated by sub-cellular redistribution, because they are targeted to intra-cellular compartments by dileucine motifs in a dynamin dependent manner. Sugar transport has been reported for GLUT6, 8, and 11; HMIT1 has been shown to be a H+/myo-inositol co-transporter. Thus, the members of the extended GLUT family exhibit a surprisingly diverse substrate specificity, and the definition of sequence elements determining this substrate specificity will require a full functional characterization of all members.

Amino acid coevolution reveals three-dimensional structure and functional domains of insect odorant receptors.

Insect odorant receptors (ORs) comprise an enormous protein family that translates environmental chemical signals into neuronal electrical activity. These heptahelical receptors are proposed to function as ligand-gated ion channels and/or to act metabotropically as G protein-coupled receptors (GPCRs). Resolving their signalling mechanism has been hampered by the lack of tertiary structural information and primary sequence similarity to other proteins. We use amino acid evolutionary covariation across these ORs to define restraints on structural proximity of residue pairs, which permit de novo generation of three-dimensional models. The validity of our analysis is supported by the location of functionally important residues in highly constrained regions of the protein. Importantly, insect OR models exhibit a distinct transmembrane domain packing arrangement to that of canonical GPCRs, establishing the structural unrelatedness of these receptor families. The evolutionary couplings and models predict odour binding and ion conduction domains, and provide a template for rationale structure-activity dissection.

The InterPro database, an integrated documentation resource for protein families, domains and functional sites.

Signature databases are vital tools for identifying distant relationships in novel sequences and hence for inferring protein function. InterPro is an integrated documentation resource for protein families, domains and functional sites, which amalgamates the efforts of the PROSITE, PRINTS, Pfam and ProDom database projects. Each InterPro entry includes a functional description, annotation, literature references and links back to the relevant member database(s). Release 2.0 of InterPro (October 2000) contains over 3000 entries, representing families, domains, repeats and sites of post-translational modification encoded by a total of 6804 different regular expressions, profiles, fingerprints and Hidden Markov Models. Each InterPro entry lists all the matches against SWISS-PROT and TrEMBL (more than 1,000,000 hits from 462,500 proteins in SWISS-PROT and TrEMBL). The database is accessible for text- and sequence-based searches at http://www.ebi.ac.uk/interpro/. Questions can be emailed to interhelp@ebi.ac.uk.

Small RNAs as regulators of primary and secondary metabolism in Pseudomonas species.

Small RNAs (sRNAs) exert important functions in pseudomonads. Classical sRNAs comprise the 4.5S, 6S, 10Sa and 10Sb RNAs, which are known in enteric bacteria as part of the signal recognition particle, a regulatory component of RNA polymerase, transfer-messenger RNA (tmRNA) and the RNA component of RNase P, respectively. Their homologues in pseudomonads are presumed to have analogous functions. Other sRNAs of pseudomonads generally have little or no sequence similarity with sRNAs of enteric bacteria. Numerous sRNAs repress or activate the translation of target mRNAs by a base-pairing mechanism. Examples of this group in Pseudomonas aeruginosa are the iron-repressible PrrF1 and PrrF2 sRNAs, which repress the translation of genes encoding iron-containing proteins, and PhrS, an anaerobically inducible sRNA, which activates the expression of PqsR, a regulator of the Pseudomonas quinolone signal. Other sRNAs sequester RNA-binding proteins that act as translational repressors. Examples of this group in P. aeruginosa include RsmY and RsmZ, which are central regulatory elements in the GacS/GacA signal transduction pathway, and CrcZ, which is a key regulator in the CbrA/CbrB signal transduction pathway. These pathways largely control the extracellular activities (including virulence traits) and the selection of the energetically most favourable carbon sources, respectively, in pseudomonads.

NS2 Proteins of GB Virus B and Hepatitis C Virus Share Common Protease Activities and Membrane Topologies.

GB virus B (GBV-B), which is hepatotropic in experimentally infected small New World primates, is a member of the Hepacivirus genus but phylogenetically relatively distant from hepatitis C virus (HCV). To gain insights into the role and specificity of hepaciviral nonstructural protein 2 (NS2), which is required for HCV polyprotein processing and particle morphogenesis, we investigated whether NS2 structural and functional features are conserved between HCV and GBV-B. We found that GBV-B NS2, like HCV NS2, has cysteine protease activity responsible for cleavage at the NS2/NS3 junction, and we experimentally confirmed the location of this junction within the viral polyprotein. A model for GBV-B NS2 membrane topology was experimentally established by determining the membrane association properties of NS2 segments fused to green fluorescent protein (GFP) and their nuclear magnetic resonance structures using synthetic peptides as well as by applying an N-glycosylation scanning approach. Similar glycosylation studies confirmed the HCV NS2 organization. Together, our data show that despite limited amino acid sequence similarity, GBV-B and HCV NS2 proteins share a membrane topology with 3 N-terminal transmembrane segments, which is also predicted to apply to other recently discovered hepaciviruses. Based on these data and using trans-complementation systems, we found that intragenotypic hybrid NS2 proteins with heterologous N-terminal membrane segments were able to efficiently trans-complement an assembly-deficient HCV mutant with a point mutation in the NS2 C-terminal domain, while GBV-B/HCV or intergenotypic NS2 chimeras were not. These studies indicate that virus- and genotype-specific intramolecular interactions between N- and C-terminal domains of NS2 are critically involved in HCV morphogenesis. IMPORTANCE: Nonstructural protein 2 (NS2) of hepatitis C virus (HCV) is a multifunctional protein critically involved in polyprotein processing and virion morphogenesis. To gain insights into NS2 mechanisms of action, we investigated whether NS2 structural and functional features are conserved between HCV and GB virus B (GBV-B), a phylogenetically relatively distant primate hepacivirus. We showed that GBV-B NS2, like HCV NS2, carries cysteine protease activity. We experimentally established a model for GBV-B NS2 membrane topology and demonstrated that despite limited sequence similarity, GBV-B and HCV NS2 share an organization with three N-terminal transmembrane segments. We found that the role of HCV NS2 in particle assembly is genotype specific and relies on critical interactions between its N- and C-terminal domains. This first comparative analysis of NS2 proteins from two hepaciviruses and our structural predictions of NS2 from other newly identified mammal hepaciviruses highlight conserved key features of the hepaciviral life cycle.

Evolutionary simulations to detect functional lineage-specific genes.

MOTIVATION: Supporting the functionality of recent duplicate gene copies is usually difficult, owing to high sequence similarity between duplicate counterparts and shallow phylogenies, which hamper both the statistical and experimental inference. RESULTS: We developed an integrated evolutionary approach to identify functional duplicate gene copies and other lineage-specific genes. By repeatedly simulating neutral evolution, our method estimates the probability that an ORF was selectively conserved and is therefore likely to represent a bona fide coding region. In parallel, our method tests whether the accumulation of non-synonymous substitutions reveals signatures of selective constraint. We show that our approach has high power to identify functional lineage-specific genes using simulated and real data. For example, a coding region of average length (approximately 1400 bp), restricted to hominoids, can be predicted to be functional in approximately 94-100% of cases. Notably, the method may support functionality for instances where classical selection tests based on the ratio of non-synonymous to synonymous substitutions fail to reveal signatures of selection. Our method is available as an automated tool, ReEVOLVER, which will also be useful to systematically detect functional lineage-specific genes of closely related species on a large scale. AVAILABILITY: ReEVOLVER is available at http://www.unil.ch/cig/page7858.html.

Low rates of X-Y recombination, not turnovers, account for homomorphic sex chromosomes in several diploid species of Palearctic green toads (Bufo viridis subgroup).

Contrasting with birds and mammals, most ectothermic vertebrates present homomorphic sex chromosomes, which might be due either to a high turnover rate or to occasional X-Y recombination. We tested these two hypotheses in a group of Palearctic green toads that diverged some 3.3 million years ago. Using sibship analyses of sex-linked markers, we show that all four species investigated share the same pair of sex chromosomes and a pattern of male heterogamety with drastically reduced X-Y recombination in males. Phylogenetic analyses of sex-linked sequences show that X and Y alleles cluster by species, not by gametolog. We conclude that X-Y homomorphy and fine-scale sequence similarity in these species do not stem from recent sex-chromosome turnovers, but from occasional X-Y recombination.

A functional and structural study of the major metalloprotease secreted by the pathogenic fungus Aspergillus fumigatus.

Fungalysins are secreted fungal peptidases with the ability to degrade the extracellular matrix proteins elastin and collagen and are thought to act as virulence factors in diseases caused by fungi. Fungalysins constitute a unique family among zinc-dependent peptidases that bears low sequence similarity to known bacterial peptidases of the thermolysin family. The crystal structure of the archetype of the fungalysin family, Aspergillus fumigatus metalloprotease (AfuMep), has been obtained for the first time. The 1.8 Å resolution structure of AfuMep corresponds to that of an autoproteolyzed proenzyme with separate polypeptide chains corresponding to the N-terminal prodomain in a binary complex with the C-terminal zinc-bound catalytic domain. The prodomain consists of a tandem of cystatin-like folds whose C-terminal end is buried into the active-site cleft of the catalytic domain. The catalytic domain harbouring the key catalytic zinc ion and its ligands, two histidines and one glutamic acid, undergoes a conspicuous rearrangement of its N-terminal end during maturation. One key positively charged amino-acid residue and the C-terminal disulfide bridge appear to contribute to its structural-functional properties. Thus, structural, biophysical and biochemical analysis were combined to provide a deeper comprehension of the underlying properties of A. fumigatus fungalysin, serving as a framework for the as yet poorly known metallopeptidases from pathogenic fungi.

The SLC2 family of facilitated hexose and polyol transporters.

The SLC2 family of glucose and polyol transporters comprises 13 members, the glucose transporters (GLUT) 1-12 and the H(+)- myo-inositol cotransporter (HMIT). These proteins all contain 12 transmembrane domains with both the amino and carboxy-terminal ends located on the cytoplasmic side of the plasma membrane and a N-linked oligosaccharide side-chain located either on the first or fifth extracellular loop. Based on sequence comparison, the GLUT isoforms can be grouped into three classes: class I comprises GLUT1-4; class II, GLUT6, 8, 10, and 12 and class III, GLUT5, 7, 9, 11 and HMIT. Despite their sequence similarity and the presence of class-specific signature sequences, these transporters carry various hexoses and HMIT is a H(+)/ myo-inositol co-transporter. Furthermore, the substrate transported by some isoforms has not yet been identified. Tissue- and cell-specific expression of the well-characterized GLUT isoforms underlies their specific role in the control of whole-body glucose homeostasis. Numerous studies with transgenic or knockout mice indeed support an important role for these transporters in the control of glucose utilization, glucose storage and glucose sensing. Much remains to be learned about the transport functions of the recently discovered isoforms (GLUT6-13 and HMIT) and their physiological role in the metabolism of glucose, myo-inositol and perhaps other substrates.

Activation mechanisms of the protease MALT1 and cleavage of one of its targets - the ribonuclease Regnase-1- in lymphocytes and lymphoma cell lines

Persistent infection induces an adaptive immune response that is mediated by T and B lymphocytes. Upon triggering with an antigen, these cells become activated and turn into fast expanding cells able to efficiently defend the host. Lymphocyte activation is controlled by a complex composed of CARMA1, BCL10 and MALT1 which regulates the NF-KB signaling pathway upon antigen triggering. Abnormally high expression or activity of either one of these three proteins can favor the development of lymphomas, while genetic defects in the pathway are associated with immunodeficiency. MALT1 was identified as a paracaspase sharing homology with other cysteine proteases, namely caspases and metacaspases. In order to be active, caspases need to dimerize. Based on their sequence similarity with MALT1, we hypothesized that dimerization might also be a mechanism of activation employed by MALT1. To address this assumption, we performed a bioinformatics modelling based on the crystal structures of several caspases. Our model suggested that the MALT1 caspase-like domain can indeed form dimers. This finding was later confirmed by several published crystal structures of MALT1. In the dimer interface of our model, we noticed the presence of charged amino acids that could potentially form salt bridges and thereby hold both monomers together. Mutation of one of these residues, E549, into alanine completely blocked the catalytic activity of MALT1. Additionally, we provided evidence for a role of E549 in promoting the MALTl-dependent growth of cells derived from diffuse large B cell lymphoma (DLBCL) of the aggressive B cell-like type (ABC). To our initial surprise, the E549A mutation showed only a partial defect in dimerization, indicating that additional residues are essential to form a stable dimer. The MALT1 crystal structures revealed a key function for E549 in stabilizing the catalytic site of the protease via its interaction with an arginine which is located next to the catalytic active cysteine. In an additional study, we discovered that MALT1 monoubiquitination is required for the catalytic activity of the protease. Interestingly, we found that the MALT1 dimer interface mutant E549A could not be monoubiquitinated. Based on these findings, we suggest that correct formation of the dimer interface is a prerequisite for monoubiquitination. In a second project, we discovered a novel target of the protease MALT1, the ribonuclease Regnase¬la It was described that the RNase activity of Regnase-1 negatively regulates immune responses. We could show that in ABC DLBCL cell lines, Regnase-1 is not only cleaved by MALT1 but also phosphorylated, at least in part, by the inhibitor of KB kinase (IKK). Both regulations appear to restrain the RNase function of Regnase-1 and thereby allow the production of pro-survival proteins. In conclusion, our studies further highlight and explain the importance of the catalytic activity of MALT1 for the activation of lymphocytes and provide additional knowledge for the development of specific drugs targeting the catalytic activity of MALT1 for immunomodulation and treatment of lymphomas.  SUMMARY IN FRENCH PhD Thesis Katrin Cabalzar 2 SUMMARY IN FRENCH Une infection persistante induit une réponse immunitaire adaptative par l'intermédiaire des lymphocytes T et B. Quand elles reconnaissent l'antigène, ces cellules sont activées et se multiplient très rapidement pour défendre efficacement l'hôte. L'activation des lymphocytes est transmise par un complexe composé de trois protéines, CARMA1, BCL10 et MALT1, qui régule la voie de signalisation NF-KB lorsque l'antigène est reconnu. L'expression ou l'activité anormalement élevée de l'une de ces trois protéines peut favoriser le développement de lymphomes, tandis que des défauts génétiques de cette voie de signalisation sont associés à l'immunodéficience. MALT1 a été identifiée comme étant une paracaspase qui partage des séquences homologues avec d'autres protéases à cystéine, comme les caspases et les métacaspases. Pour être actives, les caspases ont besoin de dimériser. Etant donné leur similarité de séquence avec MALT1, nous avons supposé que la dimérisation pouvait aussi être un mécanisme d'activation utilisé par MALT1. Pour vérifier cette hypothèse, nous avons conçu un modèle bioinformatique à partir des structures cristallographiques de plusieurs caspases. Et notre modèle a suggéré que le domaine catalytique de MALT1 était effectivement capable de former des dimères. Cette découverte a été confirmée plus tard par des publications qui montrent des structures cristallographiques dimériques de MALT1. Dans l'interface du dimère de notre modèle, nous avons remarqué la présence d'acides aminés chargés qui pouvaient former des liaisons ioniques et ainsi réunir les deux monomères. La mutation de l'un de ces résidus, E549, pour une alanine, a complètement inhibé l'activité catalytique de MALT1. De plus, nous avons mis en évidence un rôle d'E549 dans la croissance dépendante de MALT1, des cellules dérivées de lymphomes B diffus à grandes cellules (DLBCL) de sous-type cellules B actives (ABC). Dans un premier temps nous avons été surpris de constater que cette mutation révélait seulement un défaut partiel de dimérisation, ce qui indique que des acides aminés supplémentaires sont indispensables pour former un dimère stable. Les structures cristallographiques de MALT1 ont révélé un rôle primordial d'E549 dans la stabilisation du site catalytique de la protéase via son interaction avec une arginine qui se trouve à côté de la cystéine du site actif. Dans une autre étude, nous avons découvert que la monoubiquitination de MALT1 est requise pour l'activité catalytique de la protéase. A remarquer que nous avons trouvé que le mutant E549A de l'interface dimère de MALT1 n'a pas pu être monoubiquitiné. Sur la base de ces résultats, nous suggérons que la formation correcte de l'interface du dimère est une condition préalable pour la monoubiquitination. Dans un second projet, nous avons découvert une nouvelle cible de la protéase MALT1, la ribonucléase Regnase-1. Il a été décrit que l'activité RNase de Regnase-1 régulait négativement les réponses immunitaires. Nous avons pu montrer que dans les lignées cellulaires ABC DLBCL, la Regnase-1 n'était pas seulement clivée par MALT1 mais également phosphorylée, au moins en partie, par la kinase de l'inhibiteur de KB (IKK). Les deux régulations semblent supprimer la fonction RNase de Regnase-1 et permettre ainsi la stabilisation de certains ARN messagers et la production de protéines favorisant la survie. En conclusion, nos études mettent en évidence le rôle-clé de la dimérisation de MALT1 et expliquent l'importance de l'activité catalytique de MALT1 pour l'activation des lymphocytes. Ainsi, nos résultats apportent des connaissances supplémentaires pour le développement de médicaments spécifiques ciblant l'activité catalytique de MALT1, qui pourraient être utiles pour modifier les réponses immunitaires et traiter des lymphomes.

OpenFluDB, a database for human and animal influenza virus.

Although research on influenza lasted for more than 100 years, it is still one of the most prominent diseases causing half a million human deaths every year. With the recent observation of new highly pathogenic H5N1 and H7N7 strains, and the appearance of the influenza pandemic caused by the H1N1 swine-like lineage, a collaborative effort to share observations on the evolution of this virus in both animals and humans has been established. The OpenFlu database (OpenFluDB) is a part of this collaborative effort. It contains genomic and protein sequences, as well as epidemiological data from more than 27,000 isolates. The isolate annotations include virus type, host, geographical location and experimentally tested antiviral resistance. Putative enhanced pathogenicity as well as human adaptation propensity are computed from protein sequences. Each virus isolate can be associated with the laboratories that collected, sequenced and submitted it. Several analysis tools including multiple sequence alignment, phylogenetic analysis and sequence similarity maps enable rapid and efficient mining. The contents of OpenFluDB are supplied by direct user submission, as well as by a daily automatic procedure importing data from public repositories. Additionally, a simple mechanism facilitates the export of OpenFluDB records to GenBank. This resource has been successfully used to rapidly and widely distribute the sequences collected during the recent human swine flu outbreak and also as an exchange platform during the vaccine selection procedure. Database URL: http://openflu.vital-it.ch.

The SLC2 (GLUT) family of membrane transporters.

GLUT proteins are encoded by the SLC2 genes and are members of the major facilitator superfamily of membrane transporters. Fourteen GLUT proteins are expressed in the human and they are categorized into three classes based on sequence similarity. All GLUTs appear to transport hexoses or polyols when expressed ectopically, but the primary physiological substrates for several of the GLUTs remain uncertain. GLUTs 1-5 are the most thoroughly studied and all have well established roles as glucose and/or fructose transporters in various tissues and cell types. The GLUT proteins are comprised of ∼500 amino acid residues, possess a single N-linked oligosaccharide, and have 12 membrane-spanning domains. In this review we briefly describe the major characteristics of the 14 GLUT family members.

Estrella lausannensis, a new star in the Chlamydiales order.

Originally, the Chlamydiales order was represented by a single family, the Chlamydiaceae, composed of several pathogens, such as Chlamydia trachomatis, Chlamydia pneumoniae, Chlamydia psittaci and Chlamydia abortus. Recently, 6 new families of Chlamydia-related bacteria have been added to the Chlamydiales order. Most of these obligate intracellular bacteria are able to replicate in free-living amoebae. Amoebal co-culture may be used to selectively isolate amoeba-resisting bacteria. This method allowed in a previous work to discover strain CRIB 30, from an environmental water sample. Based on its 16S rRNA gene sequence similarity with Criblamydia sequanensis, strain CRIB 30 was considered as a new member of the Criblamydiaceae family. In the present work, phylogenetic analyses of the genes gyrA, gyrB, rpoA, rpoB, secY, topA and 23S rRNA as well as MALDI-TOF MS confirmed the taxonomic classification of strain CRIB 30. Morphological examination revealed peculiar star-shaped elementary bodies (EBs) similar to those of C. sequanensis. Therefore, this new strain was called "Estrella lausannensis". Finally, E. lausannensis showed a large amoebal host range and a very efficient replication rate in Acanthamoeba species. Furthermore, E. lausannensis is the first member of the Chlamydiales order to grow successfully in the genetically tractable Dictyostelium discoideum, which opens new perspectives in the study of chlamydial biology.

Odorant binding proteins of the red imported fire ant, Solenopsis invicta: an example of the problems facing the analysis of widely divergent proteins.

We describe the odorant binding proteins (OBPs) of the red imported fire ant, Solenopsis invicta, obtained from analyses of an EST library and separate 454 sequencing runs of two normalized cDNA libraries. We identified a total of 18 putative functional OBPs in this ant. A third of the fire ant OBPs are orthologs to honey bee OBPs. Another third of the OBPs belong to a lineage-specific expansion, which is a common feature of insect OBP evolution. Like other OBPs, the different fire ant OBPs share little sequence similarity (∼ 20%), rendering evolutionary analyses difficult. We discuss the resulting problems with sequence alignment, phylogenetic analysis, and tests of selection. As previously suggested, our results underscore the importance for careful exploration of the sensitivity to the effects of alignment methods for data comprising widely divergent sequences.