Sequence similarity-based proteomics in insects: characterization of the larvae venom of the Brazilian moth Cerodirphia speciosa

Using a combination of tandem mass spectrometric sequencing and sequence similarity searches, we characterized the larvae venom of the moth Cerodirphia speciosa, which belongs to the Saturniidae family of the Lepidoptera order. Despite the paucity of available database sequence resources, the approach enabled us to identify 48 out of 58 attempted spots on its two-dimensional gel electrophoresis map, which represented 37 unique proteins, whereas it was only possible to identify 13 proteins by conventional non-error tolerant database searching methods. The majority of cross-species hits were made to proteins from the phylogenetically related Lepidoptera organism, the silk worm Bombyx mori. The protein composition of the venom suggested that envenoming by C. speciosa toxins might proceed through the contact with its hemolymph, similarly to another toxic Lepidoptera organism, Lonomia obliqua.     

The evolution of heat shock genes and expression patterns of heat shock proteins in the species from temperature contrasting habitats

Heat shock genes are the most evolutionarily ancient among the systems responsible for adaptation of organisms to a harsh environment. The encoded proteins (heat shock proteins, Hsps) represent the most important factors of adaptation to adverse environmental conditions. They serve as molecular chaperones, providing protein folding and preventing aggregation of damaged cellular proteins. Structural analysis of the heat shock genes in individuals from both phylogenetically close and very distant taxa made it possible to reveal the basic trends of the heat shock gene organization in the context of adaptation to extreme conditions. Using different model objects and nonmodel species from natural populations, it was demonstrated that modulation of the Hsps expression during adaptation to different environmental conditions could be achieved by changing the number and structural organization of heat shock genes in the genome, as well as the structure of their promoters. It was demonstrated that thermotolerant species were usually characterized by elevated levels of Hsps under normal temperature or by the increase in the synthesis of these proteins in response to heat shock. Analysis of the heat shock genes in phylogenetically distant organisms is of great interest because, on one hand, it contributes to the understanding of the molecular mechanisms of evolution of adaptogenes and, on the other hand, sheds the light on the role of different Hsps families in the development of thermotolerance and the resistance to other stress factors.     

What are archaebacteria: life's third domain or monoderm prokaryotes related to Gram-positive bacteria? A new proposal for the classification of prokaryotic organisms

The evolutionary relationship within prokaryotes is examined based on signature sequences (defined as conserved inserts or deletions shared by specific taxa) and phylogenies derived from different proteins. Archaebacteria are indicated as being monophyletic by a number of proteins related to the information transfer processes. In contrast, for several other highly conserved proteins, common signature sequences are present in archaebacteria and Gram-positive bacteria, whereas Gram-negative bacteria are indicated as being distinct. For these proteins, archaebacteria do not form a phylogenetically distinct clade but show polyphyletic branching within Gram-positive bacteria. A closer relationship of archaebacteria to Gram-positive bacteria in comparison with Gram-negative bacteria is generally seen for the majority of the available gene/protein sequences. To account for these results and the fact that both archaebacteria and Gram-positive bacteria are prokaryotes surrounded by a single cell membrane, I propose that the primary division within prokaryotes is between monoderm prokaryotes (surrounded by a single membrane) and diderm prokaryotes (i.e. all true Gram-negative bacteria containing both an inner cytoplasmic membrane and an outer membrane). This proposal is consistent with both cell morphology and signature sequences in different proteins. The monophyletic nature of archaebacteria for some genes, and their polyphyletic branching within Gram-positive bacteria as suggested by others, is critically examined, and several explanations, including derivation of archaebacteria from Gram-positive bacteria in response to antibiotic selection pressure, are proposed. Signature sequences in proteins also indicate that the low-G + C Gram-positive bacteria are phylogenetically distinct from the high-G + C Gram-positive group and that the diderm prokaryotes (i.e. Gram-negative bacteria) appear to have evolved from the latter group. Protein phylogenies and signature sequences also show that all eukaryotic cells have received significant gene contributions from both an archaebacterium and a Gram-negative eubacterium. Thus, the hypothesis that archaebacteria and eukaryotes shared a common ancestor exclusive of eubacteria is not supported. These observations provide evidence for an alternate view of the evolutionary relationship among living organisms that is different from the currently popular three-domain proposal.     

Schistosoma mansoni: cloning of antigen gene sequences in Escherichia coli

Fischer rat protective antiserum (F-2x) prepared from Schistosoma mansoni-infected rats was used to screen an adult worm cDNA library constructed in a lambda gt11 bacteriophage expression vector. This led to the isolation of several clones yielding proteins reactive with antibodies in the infection serum. Counter-screening of these clones with Wistar-Furth rat nonprotective antiserum (W-2x) enabled identification of clones either uniquely or preferentially reacting with F-2x, in addition to clones of nearly equal reactivity with both antisera. Six clones were further characterized. Five expressed beta-galactosidase/S. mansoni fusion proteins which migrated more slowly in sodium dodecyl sulfate-polyacrylamide gel electrophoresis than beta-galactosidase and all were reactive in a Western immunoblot assay. The cDNA insert sizes in the clones ranged from 150 to 900 base pairs. Rabbit antibodies prepared against fusion proteins from three of the clones recognized biosynthetically radio-labeled 4-week worm proteins of sizes 20, 38, and 70 kDa, respectively. The 20- and 38-kDa proteins were among the protein antigens uniquely recognized by the F-2x protective antiserum. These proteins are therefore candidates for protective vaccine antigens and the recombinant lambda clones are now serving as useful reagents for obtaining the corresponding nucleotide gene sequences.     

Consolidating the set of known human protein-protein interactions in preparation for large-scale mapping of the human interactome

Background  

Extensive protein interaction maps are being constructed for yeast, worm, and fly to ask how the proteins organize into pathways and systems, but no such genome-wide interaction map yet exists for the set of human proteins. To prepare for studies in humans, we wished to establish tests for the accuracy of future interaction assays and to consolidate the known interactions among human proteins.     

Curli Functional Amyloid Systems Are Phylogenetically Widespread and Display Large Diversity in Operon and Protein Structure

Escherichia coli and a few other members of the Enterobacteriales can produce functional amyloids known as curli. These extracellular fibrils are involved in biofilm formation and studies have shown that they may act as virulence factors during infections. It is not known whether curli fibrils are restricted to the Enterobacteriales or if they are phylogenetically widespread. The growing number of genome-sequenced bacteria spanning many phylogenetic groups allows a reliable bioinformatic investigation of the phylogenetic diversity of the curli system. Here we show that the curli system is phylogenetically much more widespread than initially assumed, spanning at least four phyla. Curli fibrils may consequently be encountered frequently in environmental as well as pathogenic biofilms, which was supported by identification of curli genes in public metagenomes from a diverse range of habitats. Identification and comparison of curli subunit (CsgA/B) homologs show that these proteins allow a high degree of freedom in their primary protein structure, although a modular structure of tightly spaced repeat regions containing conserved glutamine, asparagine and glycine residues has to be preserved. In addition, a high degree of variability within the operon structure of curli subunits between bacterial taxa suggests that the curli fibrils might have evolved to fulfill specific functions. Variations in the genetic organization of curli genes are also seen among different bacterial genera. This suggests that some genera may utilize alternative regulatory pathways for curli expression. Comparison of phylogenetic trees of Csg proteins and the 16S rRNA genes of the corresponding bacteria showed remarkably similar overall topography, suggesting that horizontal gene transfer is a minor player in the spreading of the curli system.     

Molecular Adaptation in the Ice Worm, <Emphasis Type="Italic">Mesenchytraeus solifugus</Emphasis>: Divergence of Energetic-Associated Genes

The ice worm, Mesenchytraeus solifugus, is the largest glacially obligate metazoan and among only a few metazoan species that complete their life cycle at 0°C. We conducted a large-scale sequencing analysis of cDNAs isolated from ice worm anterior segments. Sequence comparisons among an available group of ice worm, arthropod, chordate, and nematode homologues suggest that ice worms encode proteins that are less bulky, are less polar, and contain fewer charged residues. Also, subunits of the catalytic F₁ ATP synthase complex appear to have diverged more rapidly than other ice worm genes examined, suggesting a role in cold-temperature adaptation. Modeling of F₁ ATP synthase and subunits identified nonconservative, ice worm-specific amino acid substitutions at subunit contact sites and at sites proximal to the catalytic site.Reviewing Editor: Dr. Graziano Pesole(Angela H. Farrell and Kristi A. Hohenstein) These authors contributed equally.     

Cell division genes and proteins in bacterial cells

In this review, genes and proteins involved in cytokinesis and cell proliferation of cell-wall bacteria and mycoplasms are considered. We hope that this comparative analysis of genes and proteins of phylogenetically distant bacteria, including the minimal cells of mycoplasmas, can be useful for understanding the basic principles of prokaryotic cell division. The ftsZ gene was found among representatives of all bacterial groups. The recent data indicate that FtsZ protein plays the central role in the process of bacterial cell division. FtsZ protein was revealed in all Eubacterial groups (including mycoplasmas), in Archaebacteria and chloroplasts, All FtsZ proteins are able to form protofilaments as a result of polymerization in vitro and demonstrate GTF-ase activity. On the base of these properties and some similarities in amino acid sequences with tubulins, it has been suggested that FtsZ protein is an evolutionary ancestor of Eukaryotic tubulins. On the earliest stage of bacterial cytokinesis FtsZ protein assembles into a submembranous Z-ring which encircles bacterial cell in the predivisional site. Some other bacterial proteins take part in stabilization and contraction of the Z-ring, which is considered as a cytoskeleton-like bacterial structure.     

Regulatory role for a conserved motif adjacent to the homeodomain of Hox10 proteins

Development of the vertebrate axial skeleton requires the concerted activity of several Hox genes. Among them, Hox genes belonging to the paralog group 10 are essential for the formation of the lumbar region of the vertebral column, owing to their capacity to block rib formation. In this work, we explored the basis for the rib-repressing activity of Hox10 proteins. Because genetic experiments in mice demonstrated that Hox10 proteins are strongly redundant in this function, we first searched for common motifs among the group members. We identified the presence of two small sequences flanking the homeodomain that are phylogenetically conserved among Hox10 proteins and that seem to be specific for this group. We show here that one of these motifs is required but not sufficient for the rib-repressing activity of Hox10 proteins. This motif includes two potential phosphorylation sites, which are essential for protein activity as their mutation to alanines resulted in a total loss of rib-repressing properties. Our data indicates that this motif has a significant regulatory function, modulating interactions with more N-terminal parts of the Hox protein, eventually triggering the rib-repressing program. In addition, this motif might also regulate protein activity by alteration of the protein's DNA-binding affinity through changes in the phosphorylation state of two conserved tyrosine residues within the homeodomain.     

Improved Perceptions and Practices Related to Schistosomiasis and Intestinal Worm Infections Following PHAST Intervention on Kome Island,North-Western Tanzania

Schistosomiasis and intestinal worm infections are widespread diseases of public health importance in Tanzania. A study on perceptions and practices related to schistosomiasis and intestinal worm infections was undertaken among a community population of Kome Island in Sengerema District, north-western Tanzania, where intestinal schistosomiasis and intestinal worm infections are endemic. Schistosomiasis and intestinal worm-related perceptions and practices were assessed before and 3 years after implementation of a participatory hygiene and sanitation transformation (PHAST) intervention as a control measure. Data were obtained from baseline and post-intervention knowledge, attitudes, and practices (KAP) questionnaire surveys conducted twice in 2009 and 2012 among 82 individuals aged ≥15 years. We found significant increases in respondents’ knowledge of the cause, transmission, symptoms, health consequences, and prevention of schistosomiasis and intestinal worm infections after PHAST intervention. The increase in respondents’ knowledge on almost all aspects of the said infections was translated into actions to control schistosomiasis and intestinal worm infections. This has not been achieved by chance, but due to well-designed and locally-adapted PHAST intervention. We conclude that despite criticisms, PHAST approach is still useful in empowering communities to control water, sanitation, and hygiene related infectious diseases such as schistosomiasis and intestinal worm infections.     

Cloning, expression analysis, and sequence diversity of genes encoding two different immunodominant membrane proteins in poinsettia branch-inducing phytoplasma (PoiBI)

Poinsettia branch-inducing phytoplasma (PoiBI) is a phytopathogenic bacterium that infects poinsettia, and is associated with the free-branching morphotype (characterized by many axillary shoots and flowers) of many commercially grown poinsettias. The major membrane proteins of phytoplasmas are classified into three general types, that is, immunodominant membrane protein (Imp), immunodominant membrane protein A (IdpA), and antigenic membrane protein (Amp). These membrane proteins are often used as targets for the production of antibodies used in phytoplasma detection. Herein, we cloned and sequenced the imp and idpA genes of PoiBI strains from 26 commercial poinsettia cultivars. Although the amino acid sequences of the encoded IdpA proteins were invariant, those of the encoded Imp varied among the PoiBI isolates, with no synonymous nucleotide substitution. Western blotting and immunohistochemical analyses revealed that the amount of Imp expressed exceeded that of IdpA, in contrast to the case of a related phytoplasma-disease, western X-disease, for which the major membrane protein appears to be IdpA, not Imp. These results suggest that even phylogenetically close phytoplasmas express different types of major membrane proteins.     

Gene arrangements characteristic of the phylum Actinobacteria

The availability of genomic sequence data allows new challenges to various biological problems. One of such attempts is the extraction of phylogenetic information from gene order data of genomes. Phylogenetic inferences are most commonly carried out on the basis of 16S rRNA trees, which sometimes produce unresolved or unreliable branching orders. One example for such a low resolution is recognized in the branching pattern among the phylum Actinobacteria. Here, gene arrangements characteristic of the Actinobacteria were identified, based on which Symbiobacterium thermophilum is phylogenetically placed outside that phylum, this being in contrast to 16S rRNA trees and to the current taxonomy in GenBank. Three transposition suggestive arrangements were found which support a notion that Rubrobacter xylanophilus is the earliest diverging species among the completely sequenced Actinobacteria. The gene arrangements identified here serve as a complement to previously reported indels and proteins characteristic of this phylum.     

Interactome analysis of Caenorhabditis elegans synapses by TurboID-based proximity labeling

Proximity labeling provides a powerful in vivo tool to characterize the proteome of subcellular structures and the interactome of specific proteins. The nematode Caenorhabditis elegans is one of the most intensely studied organisms in biology, offering many advantages for biochemistry. Using the highly active biotin ligase TurboID, we optimize here a proximity labeling protocol for C. elegans. An advantage of TurboID is that biotin''s high affinity for streptavidin means biotin-labeled proteins can be affinity-purified under harsh denaturing conditions. By combining extensive sonication with aggressive denaturation using SDS and urea, we achieved near-complete solubilization of worm proteins. We then used this protocol to characterize the proteomes of the worm gut, muscle, skin, and nervous system. Neurons are among the smallest C. elegans cells. To probe the method''s sensitivity, we expressed TurboID exclusively in the two AFD neurons and showed that the protocol could identify known and previously unknown proteins expressed selectively in AFD. The active zones of synapses are composed of a protein matrix that is difficult to solubilize and purify. To test if our protocol could solubilize active zone proteins, we knocked TurboID into the endogenous elks-1 gene, which encodes a presynaptic active zone protein. We identified many known ELKS-1-interacting active zone proteins, as well as previously uncharacterized synaptic proteins. Versatile vectors and the inherent advantages of using C. elegans, including fast growth and the ability to rapidly make and functionally test knock-ins, make proximity labeling a valuable addition to the armory of this model organism.     

The behavior of parasitic flatworms in vivo: what is the role of the brain?

The ecological interactions that contribute to successful host-parasite relationships are complex and involve all levels of biotic organization between the participants. At the level of parasites living within their hosts, it is felt that the parasite's environment is predictable because of host mechanisms maintaining biochemical and physiological homeostasis. It is hypothesized that fixed behavior patterns in the parasites will evolve under these specialized conditions. Current thinking on fixed behaviors in invertebrates holds that they are generated by specialized neural circuits in the brain. Therefore, it can be expected that the brains of parasitic flatworms will have important roles in the control of the organisms' behaviors. However, in the tapeworm Hymenolepis diminuta, complex fixed patterns of behavior, associated with locomotion and migration, are not affected by the removal of the worm's brain. This suggests peripheral, and not central, control of fixed behaviors. In Fasciola hepatica, at least 6 distinct fixed patterns of behavior are responsible for guiding the worm to its final habitat in the liver. Giant neurons and other phylogenetically advanced features develop in the adult worm's brain after the expression of the sequence of distinct migration behaviors. Yet, there is no apparent new locomotory behavior, corresponding to the new advanced brain, as the parasite assumes its placid life-style as a hematophage in the bile duct. Removal of the adult brain of this parasite also does not appear to affect worm locomotory activity. Thus, the regulation and control of locomotion may not be the only important roles for the brains of parasitic flatworms. It is suggested that neuroethological approaches may hold the key to understanding the biology of these parasites.     

The splicing factor U2AF65 is functionally conserved in the thermotolerant deep-sea worm Alvinella pompejana

Due to their inherent stability, thermophilic bacteria and archaea serve as important resources for biochemical and biophysical analyses of many biological processes. Unfortunately, scientists characterizing eukaryote-specific processes, such as nuclear pre-mRNA splicing, are unable to take advantage of these sources of thermostable proteins. To identify and provide a source of thermostable eukaryotic proteins, we are characterizing splicing factors in the thermotolerant deep-sea vent polychaete, Alvinella pompejana. This worm, also known as the Pompeii worm, is found in the extreme environment of deep-sea hydrothermal vents, and is one of the most thermotolerant eukaryotic organisms known. We report on detailed analyses of U2AF65, the large subunit of the U2 small nuclear ribonucleoprotein auxiliary factor, an essential splicing factor important for intron definition and alternative splicing. The cloning and characterization of Pompeii U2AF65 show it is highly similar to human U2AF65 in sequence and function and is more thermostable than the human protein when bound to RNA in vitro. Notably, Pompeii U2AF65 can restore splicing in a human extract depleted of human U2AF. We also determine that the general splicing mechanisms and signal sequences are conserved in the Pompeii worm, an annelid which has previously been uncharacterized in terms of splicing factors and signals.     

Where have all the interactions gone? Estimating the coverage of two-hybrid protein interaction maps

Yeast two-hybrid screens are an important method for mapping pairwise physical interactions between proteins. The fraction of interactions detected in independent screens can be very small, and an outstanding challenge is to determine the reason for the low overlap. Low overlap can arise from either a high false-discovery rate (interaction sets have low overlap because each set is contaminated by a large number of stochastic false-positive interactions) or a high false-negative rate (interaction sets have low overlap because each misses many true interactions). We extend capture-recapture theory to provide the first unified model for false-positive and false-negative rates for two-hybrid screens. Analysis of yeast, worm, and fly data indicates that 25% to 45% of the reported interactions are likely false positives. Membrane proteins have higher false-discovery rates on average, and signal transduction proteins have lower rates. The overall false-negative rate ranges from 75% for worm to 90% for fly, which arises from a roughly 50% false-negative rate due to statistical undersampling and a 55% to 85% false-negative rate due to proteins that appear to be systematically lost from the assays. Finally, statistical model selection conclusively rejects the Erd?s-Rényi network model in favor of the power law model for yeast and the truncated power law for worm and fly degree distributions. Much as genome sequencing coverage estimates were essential for planning the human genome sequencing project, the coverage estimates developed here will be valuable for guiding future proteomic screens. All software and datasets are available in and , -, and -, and are also available from our Web site, http://www.baderzone.org.     

Weshalb hemmen Würmer Allergien? Parasiten als Immunologen

Parasitic worms survive within their immunocompetent hosts by modulating their immune system and by inhibiting inflammatory responses directed against the parasites. This immunomodulation has a spill over effect and also inhibits inflammatory responses originating from other causes. For this reason, persons who are infected with certain species of worms show a lower rate of allergic diseases as compared to persons who are free of parasites. In the same line, studies in mouse models revealed that many inflammatory diseases can be treated by worm infections. This effect is among others owing to specific proteins that are released by the worms. Such secreted immunomodulators, shaped by co‐evolution between parasites and their hosts, could become lead compounds for the development of new therapies against allergic and inflammatory diseases.     

CPF and CPFL, two related gene families encoding cuticular proteins of Anopheles gambiae and other insects

Cuticular proteins (CPs) are structural proteins of insects as well as other arthropods. Several CP families have been described, among them a small family defined by a 51 amino acid motif [Andersen, S.O., Rafn, K., Roepstorff, P., 1997. Sequence studies of proteins from larval and pupal cuticle of the yellow meal worm, Tenebrio molitor. Insect Biochem. Mol. Biol. 27, 121-131]. We identified four proteins of this family in Anopheles gambiae that we have named CPF. We have also identified CPFs from other insects by searching databases. Alignment of these CPF proteins showed that the conserved region is only 44 aa long and revealed another conserved motif at the C-terminus. A dendrogram divided the CPF proteins into four groups, one basal and three specialized. We also identified several proteins of another CP family, CPFL, which has similarities to CPFs. CPFs and CPFLs share some protein motifs. Expression studies with real-time qRT-PCR of the A. gambiae CPFs and CPFLs showed that the four CPFs and one CPFL gene are expressed just before pupal or adult ecdysis, suggesting that they are components of the outer layer of pupal and adult cuticles. The other CPFLs appear to contribute to larval cuticle. Recombinant CPF proteins did not bind to chitin in the assay we used.     

Sphingosine-1-Phosphate Metabolism and Intestinal Tumorigenesis: Lipid Signaling Strikes Again

Superficially similar traits in phylogenetically unrelated species often result from adaptation to common selection pressures. Examples of convergent evolution are known at the levels of whole organisms, organ systems, gene networks and specific proteins. The phenotypic properties of living things, on the other hand, are determined in large part by complex networks of interacting proteins. Here we present a mathematical model of the network of proteins that controls DNA synthesis and cell division in the alpha-proteobacterium, Caulobacter crescentus. By comparing the protein regulatory circuits for cell reproduction in Caulobacter with that in budding yeast (Saccharomyces cerevisiae), we suggest that convergent evolution may have created similar molecular reaction networks in order to accomplish the same purpose of coordinating DNA synthesis to cell division. Although the genes and proteins involved in cell cycle regulation in prokaryotes and eukaryotes are very different and (apparently) phylogenetically unrelated, they seem to be wired together in similar regulatory networks, which coordinate cell cycle events by identical dynamical principles.     

RCC1-like repeat proteins: a pangenomic, structurally diverse new superfamily of beta-propeller domains

The beta-propeller fold is a phylogenetically widespread, common protein architecture able to support a range of different functions such as catalysis, ligand binding and transport, regulation and protein binding. Interestingly, it appears that the beta-propeller topology is also compatible with strikingly diverse sequences. Amongst this diversity, there are three large groups of proteins with related sequences and very important cellular and intercellular regulatory functions: WD, kelch, and YWTD proteins. A common characteristic between these protein families is that their sequences, while distinct, all contain internal repeats 40-45 residues long. Through a pangenomic analysis using internal repeat profiles derived from the structurally known propeller modules of the eukaryotic protein RCC1 and the related prokaryotic protein BLIP-II, we have defined a new superfamily of propeller repeats, the RCC1-like repeats (RLRs). These sequences turn out to be more phylogenetically widespread than other large groups of propeller proteins, occurring in both prokaryotic and eukaryotic genomes. Interestingly, our research showed that RLR domains with different numbers of repeats exist, ranging from 3 to 7, and possibly more. A novel, intriguing finding is the discovery of sequences with 3 repeats, as well as proteins with 10 modular units, though in the latter case it is not clear whether these are made of two 5-bladed domains or a single, novel 10-bladed propeller. In addition, the results indicate that circular permutation events may have taken place in the evolution of these proteins. It is now established that the group of RLR proteins is extremely numerous and is characterized by unique, remarkable features which place it in a position of special interest as an important superfamily of proteins in nature.