An integrated transcriptomic and proteomic approach to identify the main Torymus sinensis venom components.

During oviposition, ectoparasitoid wasps not only inject their eggs but also a complex mixture of proteins and peptides (venom) in order to regulate the host physiology to benefit their progeny. Although several endoparasitoid venom proteins have been identified, little is known about the components of ectoparasitoid venom. To characterize the protein composition of Torymus sinensis Kamijo (Hymenoptera: Torymidae) venom, we used an integrated transcriptomic and proteomic approach and identified 143 venom proteins. Moreover, focusing on venom gland transcriptome, we selected additional 52 transcripts encoding putative venom proteins. As in other parasitoid venoms, hydrolases, including proteases, phosphatases, esterases, and nucleases, constitute the most abundant families in T. sinensis venom, followed by protease inhibitors. These proteins are potentially involved in the complex parasitic syndrome, with different effects on the immune system, physiological processes and development of the host, and contribute to provide nutrients to the parasitoid progeny. Although additional in vivo studies are needed, initial findings offer important information about venom factors and their putative host effects, which are essential to ensure the success of parasitism.

The DNases of pathogenic Lancefield streptococci.

DNases are abundant among the pathogenic streptococci, with most species harbouring genes for at least one. Despite their prevalence, however, the role for these extracellular enzymes is still relatively unclear. The DNases of the Lancefield group A Streptococcus, S. pyogenes are the best characterized, with a total of eight DNase genes identified so far. Six are known to be associated with integrated prophages. Two are chromosomally encoded, and one of these is cell-wall anchored. Homologues of both prophage-associated and chromosomally encoded S. pyogenes DNases have been identified in other streptococcal species, as well as other unique DNases. A major role identified for streptococcal DNases appears to be in the destruction of extracellular traps produced by immune cells, such as neutrophils, to ensnare bacteria and kill them. These traps are composed primarily of DNA which can be degraded by the secreted and cell-wall-anchored streptococcal DNases. DNases can also reduce TLR-9 signalling to dampen the immune response and produce cytotoxic deoxyadenosine to limit phagocytosis. Upper respiratory tract infection models of S. pyogenes have identified a role for DNases in potentiating infection and transmission, possibly by limiting the immune response or through some other unknown mechanism. Streptococcal DNases may also be involved in interacting with other microbial communities through communication, bacterial killing and disruption of competitive biofilms, or control of their own biofilm production. The contribution of DNases to pathogenesis may therefore be wide ranging and extend beyond direct interference with the host immune response.

Comprehensive classification of the PIN domain-like superfamily.

PIN-like domains constitute a widespread superfamily of nucleases, diverse in terms of the reaction mechanism, substrate specificity, biological function and taxonomic distribution. Proteins with PIN-like domains are involved in central cellular processes, such as DNA replication and repair, mRNA degradation, transcription regulation and ncRNA maturation. In this work, we identify and classify the most complete set of PIN-like domains to provide the first comprehensive analysis of sequence-structure-function relationships within the whole PIN domain-like superfamily. Transitive sequence searches using highly sensitive methods for remote homology detection led to the identification of several new families, including representatives of Pfam (DUF1308, DUF4935) and CDD (COG2454), and 23 other families not classified in the public domain databases. Further sequence clustering revealed relationships between individual sequence clusters and showed heterogeneity within some families, suggesting a possible functional divergence. With five structural groups, 70 defined clusters, over 100,000 proteins, and broad biological functions, the PIN domain-like superfamily constitutes one of the largest and most diverse nuclease superfamilies. Detailed analyses of sequences and structures, domain architectures, and genomic contexts allowed us to predict biological function of several new families, including new toxin-antitoxin components, proteins involved in tRNA/rRNA maturation and transcription/translation regulation.

Functional disruption of human leukocyte antigen II in human embryonic stem cell.

BACKGROUND: Theoretically human embryonic stem cells (hESCs) have the capacity to self-renew and differentiate into all human cell types. Therefore, the greatest promise of hESCs-based therapy is to replace the damaged tissues of patients suffering from traumatic or degenerative diseases by the exact same type of cells derived from hESCs. Allograft immune rejection is one of the obstacles for hESCs-based clinical applications. Human leukocyte antigen (HLA) II leads to CD4(+) T cells-mediated allograft rejection. Hence, we focus on optimizing hESCs for clinic application through gene modification. RESULTS: Transcription activator-like effector nucleases (TALENs) were used to target MHC class II transactivator (CIITA) in hESCs efficiently. CIITA (-/-) hESCs did not show any difference in the differentiation potential and self-renewal capacity. Dendritic cells (DCs) derived from CIITA (-/-) hESCs expressed CD83 and CD86 but without the constitutive HLA II. Fibroblasts derived from CIITA (-/-) hESCs were powerless in IFN-γ inducible expression of HLA II. CONCLUSION: We generated HLA II defected hESCs via deleting CIITA, a master regulator of constitutive and IFN-γ inducible expression of HLA II genes. CIITA (-/-) hESCs can differentiate into tissue cells with non-HLA II expression. It's promising that CIITA (-/-) hESCs-derived cells could be used in cell therapy (e.g., T cells and DCs) and escape the attack of receptors' CD4(+) T cells, which are the main effector cells of cellular immunity in allograft.

Functional disruption of human leukocyte antigen II in human embryonic stem cell

BACKGROUND: Theoretically human embryonic stem cells (hESCs) have the capacity to self-renew and differentiate into all human cell types. Therefore, the greatest promise of hESCs-based therapy is to replace the damaged tissues of patients suffering from traumatic or degenerative diseases by the exact same type of cells derived from hESCs. Allo-graft immune rejection is one of the obstacles for hESCs-based clinical applications. Human leukocyte antigen (HLA) II leads to CD4+ T cells-mediated allograft rejection. Hence, we focus on optimizing hESCs for clinic application through gene modification. RESULTS: Transcription activator-like effector nucleases (TALENs) were used to target MHC class II transactivator (CIITA) in hESCs efficiently. CIITA(-/-)hESCs did not show any difference in the differentiation potential and self-renewal capacity. Dendritic cells (DCs) derived from CIITA(-/-)hESCs expressed CD83 and CD86 but without the constitutive HLA II. Fibroblasts derived from CIITA(-/-)hESCs were powerless in IFN-γ inducible expression of HLA II. CONCLUSION: We generated HLA II defected hESCs via deleting CIITA, a master regulator of constitutive and IFN-γ inducible expression of HLA II genes. CIITA(-/-)hESCs can differentiate into tissue cells with non-HLA II expression. It's promising that CIITA(-/-)hESCs-derived cells could be used in cell therapy (e.g., T cells and DCs) and escape the attack of receptors' CD4+ T cells, which are the main effector cells of cellular immunity in allograft.

Is crab duplex-specific nuclease a member of the Serratia family of non-specific nucleases?

Kamchatka crab duplex-specific nuclease (Par_DSN) has been classified as a member of the family of DNA/RNA non-specific beta-beta-alpha metal finger (bba-Me-finger) nucleases, the archetype of which is the nuclease from Serratia marcescens. Although the enzyme under investigation seems to belong to the family of S. marcescens nucleases, Par_DSN exhibits a marked preference for double-stranded DNA as a substrate and this property is unusual for other members of this family. We have searched other Arthropod species and identified a number of novel Par_DSN homologs. A phylogenetic analysis demonstrates that the Par_DSN-like enzymes constitute a separate branch in the evolutionary tree of bba-Me-finger nucleases. Combining sequence analysis and site-directed mutagenesis, we found that Par_DSN and its homologs possess the nuclease domain that is slightly longer than that of classic Serratia relatives. The active site composition of Par_DSN is similar but not identical to that of classic Serratia nucleases. Based on these findings, we proposed a new classification of Par_DSN-like nucleases.

Molecular and functional analyses of a novel class I secretory nuclease from the human pathogen, Leishmania donovani.

The primitive protozoan pathogen of humans, Leishmania donovani, resides and multiplies in highly restricted micro-environments within their hosts (i.e. as promastigotes in the gut lumen of their sandfly vectors and as amastigotes in the phagolysosomal compartments of infected mammalian macrophages). Like other trypanosomatid parasites, they are purine auxotrophs (i.e. lack the ability to synthesize purines de novo) and therefore are totally dependent upon salvaging these essential nutrients from their hosts. In that context, in this study we identified a unique 35-kDa, dithiothreitol-sensitive nuclease and showed that it was constitutively released/secreted by both promastigote and amastigote developmental forms of this parasite. By using several different molecular approaches, we identified and characterized the structure of LdNuc(s), a gene that encodes this new 35-kDa class I nuclease family member in these organisms. Homologous episomal expression of an epitope-tagged LdNuc(s) chimeric construct was used in conjunction with an anti-LdNuc(s) peptide antibody to delineate the functional and biochemical properties of this unique 35-kDa parasite released/secreted enzyme. Results of coupled immunoprecipitation-enzyme activity analyses demonstrated that this "secretory" enzyme could hydrolyze a variety of synthetic polynucleotides as well as several natural nucleic acid substrates, including RNA and single- and double-stranded DNA. Based on these cumulative observations, we hypothesize that within the micro-environments of its host, this leishmanial "secretory" nuclease could function at a distance away from the parasite to harness (i.e. hydrolyze/access) host-derived nucleic acids to satisfy the essential purine requirements of these organisms. Thus, this enzyme might play an important role(s) in facilitating the survival, growth, and development of this important human pathogen.

Structure and function of nucleases in DNA repair: shape, grip and blade of the DNA scissors.

DNA nucleases catalyze the cleavage of phosphodiester bonds. These enzymes play crucial roles in various DNA repair processes, which involve DNA replication, base excision repair, nucleotide excision repair, mismatch repair, and double strand break repair. In recent years, new nucleases involved in various DNA repair processes have been reported, including the Mus81 : Mms4 (Eme1) complex, which functions during the meiotic phase and the Artemis : DNA-PK complex, which processes a V(D)J recombination intermediate. Defects of these nucleases cause genetic instability or severe immunodeficiency. Thus, structural biology on various nuclease actions is essential for the elucidation of the molecular mechanism of complex DNA repair machinery. Three-dimensional structural information of nucleases is also rapidly accumulating, thus providing important insights into the molecular architectures, as well as the DNA recognition and cleavage mechanisms. This review focuses on the three-dimensional structure-function relationships of nucleases crucial for DNA repair processes.

The in vitro interaction of Streptococcus pyogenes with human pharyngeal cells induces a phage-encoded extracellular DNase.

The role lysogenic bacteriophage play in the pathogenesis of the host bacterium is poorly understood. In a previous study, we found that streptococcal coculture with human pharyngeal cells resulted in the induction of lysogenic bacteriophage as well as the phage-associated streptococcal pyrogenic exotoxin C (SpeC). In this study, we have determined that in addition to SpeC induction, a number of other streptococcal proteins are also released by the bacteria during coculture with pharyngeal cells. Among these, we identified and characterized a novel 27-kDa secreted protein. Sequence analysis of this novel protein demonstrated it to be encoded by the same lysogenic bacteriophage which harbors speC. Protein sequence analysis revealed varied homologies with several streptococcal DNases. Further biochemical characterization of the recombinantly expressed protein verified it to be a divalent cation-dependent streptococcal phage-encoded DNase (Spd1). Although functionally distinct, SpeC and Spd1 are associated by a number of parameters, including genetic proximity and transcriptional regulation. Finally, we speculate on the induction of phage-encoded DNase (Spd1) enhancing the fitness of both bacteria and phage.

SURVEY AND SUMMARY: holliday junction resolvases and related nucleases: identification of new families, phyletic distribution and evolutionary trajectories.

Holliday junction resolvases (HJRs) are key enzymes of DNA recombination. A detailed computer analysis of the structural and evolutionary relationships of HJRs and related nucleases suggests that the HJR function has evolved independently from at least four distinct structural folds, namely RNase H, endonuclease, endonuclease VII-colicin E and RusA. The endonuclease fold, whose structural prototypes are the phage lambda exonuclease, the very short patch repair nuclease (Vsr) and type II restriction enzymes, is shown to encompass by far a greater diversity of nucleases than previously suspected. This fold unifies archaeal HJRs, repair nucleases such as RecB and Vsr, restriction enzymes and a variety of predicted nucleases whose specific activities remain to be determined. Within the RNase H fold a new family of predicted HJRs, which is nearly ubiquitous in bacteria, was discovered, in addition to the previously characterized RuvC family. The proteins of this family, typified by Escherichia coli YqgF, are likely to function as an alternative to RuvC in most bacteria, but could be the principal HJRs in low-GC Gram-positive bacteria and AQUIFEX: Endonuclease VII of phage T4 is shown to serve as a structural template for many nucleases, including MCR:A and other type II restriction enzymes. Together with colicin E7, endonuclease VII defines a distinct metal-dependent nuclease fold. As a result of this analysis, the principal HJRs are now known or confidently predicted for all bacteria and archaea whose genomes have been completely sequenced, with many species encoding multiple potential HJRs. Horizontal gene transfer, lineage-specific gene loss and gene family expansion, and non-orthologous gene displacement seem to have been major forces in the evolution of HJRs and related nucleases. A remarkable case of displacement is seen in the Lyme disease spirochete Borrelia burgdorferi, which does not possess any of the typical HJRs, but instead encodes, in its chromosome and each of the linear plasmids, members of the lambda exonuclease family predicted to function as HJRs. The diversity of HJRs and related nucleases in bacteria and archaea contrasts with their near absence in eukaryotes. The few detected eukaryotic representatives of the endonuclease fold and the RNase H fold have probably been acquired from bacteria via horizontal gene transfer. The identity of the principal HJR(s) involved in recombination in eukaryotes remains uncertain; this function could be performed by topoisomerase IB or by a novel, so far undetected, class of enzymes. Likely HJRs and related nucleases were identified in the genomes of numerous bacterial and eukaryotic DNA viruses. Gene flow between viral and cellular genomes has probably played a major role in the evolution of this class of enzymes. This analysis resulted in the prediction of numerous previously unnoticed nucleases, some of which are likely to be new restriction enzymes.

Analysis of nuclear degradation during lens cell differentiation.

Lens cells demonstrate a terminal differentiation process with loss of their organelles including nuclei. Chromatin disappearance is characterised by the same changes as most apoptotic cells, i.e. condensation of chromatin and cleavage into high molecular weight fragments and oligonucleosomes. The endo-deoxyribonucleases (bicationic (Ca2+, Mg2+), mono-cationic (Ca2+ or Mg2+) and acidic non-cationic dependent nucleases) are present in lens fibre cells. Our results suggest that the acidic non-cationic nuclease (DNase II) plays a major role in chromatin cleavage. This nuclease, known to be lysosomal, is found in lens fibre nuclei and only an antibody directed against DNase II inhibits the acidic DNA cleavage of lens fibre nuclei. In addition, there must be another DNase implicated in the process which is not DNase I but appears to be a Ca2+, Mg2+ dependent molecule. Regulation of these DNase activities may be accomplished by the effect of post-translational modifications, acidic pH, mitochondrial release molecules, growth factors or oncogenes. Finally, fibre cells lose organelles without cytoplasmic elimination. The survival of these differentiated cells might be due to the action of survival factors such as FGF 1.