Novel Hydrophobins from <Emphasis Type="Italic">Trichoderma</Emphasis> Define a New Hydrophobin Subclass: Protein Properties,Evolution, Regulation and Processing

Hydrophobins are small proteins, characterised by the presence of eight positionally conserved cysteine residues, and are present in all filamentous asco- and basidiomycetes. They are found on the outer surfaces of cell walls of hyphae and conidia, where they mediate interactions between the fungus and the environment. Hydrophobins are conventionally grouped into two classes (class I and II) according to their solubility in solvents, hydropathy profiles and spacing between the conserved cysteines. Here we describe a novel set of hydrophobins from Trichoderma spp. that deviate from this classification in their hydropathy, cysteine spacing and protein surface pattern. Phylogenetic analysis shows that they form separate clades within ascomycete class I hydrophobins. Using T. atroviride as a model, the novel hydrophobins were found to be expressed under conditions of glucose limitation and to be regulated by differential splicing.     

N-Terminal Presequence-Independent Import of Phosphofructokinase into Hydrogenosomes of Trichomonas vaginalis

Mitochondrial evolution entailed the origin of protein import machinery that allows nuclear-encoded proteins to be targeted to the organelle, as well as the origin of cleavable N-terminal targeting sequences (NTS) that allow efficient sorting and import of matrix proteins. In hydrogenosomes and mitosomes, reduced forms of mitochondria with reduced proteomes, NTS-independent targeting of matrix proteins is known. Here, we studied the cellular localization of two glycolytic enzymes in the anaerobic pathogen Trichomonas vaginalis: PP_i-dependent phosphofructokinase (TvPP_i-PFK), which is the main glycolytic PFK activity of the protist, and ATP-dependent PFK (TvATP-PFK), the function of which is less clear. TvPP_i-PFK was detected predominantly in the cytosol, as expected, while all four TvATP-PFK paralogues were imported into T. vaginalis hydrogenosomes, although none of them possesses an NTS. The heterologous expression of TvATP-PFK in Saccharomyces cerevisiae revealed an intrinsic capability of the protein to be recognized and imported into yeast mitochondria, whereas yeast ATP-PFK resides in the cytosol. TvATP-PFK consists of only a catalytic domain, similarly to “short” bacterial enzymes, while ScATP-PFK includes an N-terminal extension, a catalytic domain, and a C-terminal regulatory domain. Expression of the catalytic domain of ScATP-PFK and short Escherichia coli ATP-PFK in T. vaginalis resulted in their partial delivery to hydrogenosomes. These results indicate that TvATP-PFK and the homologous ATP-PFKs possess internal structural targeting information that is recognized by the hydrogenosomal import machinery. From an evolutionary perspective, the predisposition of ancient ATP-PFK to be recognized and imported into hydrogenosomes might be a relict from the early phases of organelle evolution.     

Reticulate evolution in diploid and tetraploid species of Polystachya (Orchidaceae) as shown by plastid DNA sequences and low-copy nuclear genes

Background and Aims

Here evidence for reticulation in the pantropical orchid genus Polystachya is presented, using gene trees from five nuclear and plastid DNA data sets, first among only diploid samples (homoploid hybridization) and then with the inclusion of cloned tetraploid sequences (allopolyploids). Two groups of tetraploids are compared with respect to their origins and phylogenetic relationships.

Methods

Sequences from plastid regions, three low-copy nuclear genes and ITS nuclear ribosomal DNA were analysed for 56 diploid and 17 tetraploid accessions using maximum parsimony and Bayesian inference. Reticulation was inferred from incongruence between gene trees using supernetwork and consensus network analyses and from cloning and sequencing duplicated loci in tetraploids.

Key Results

Diploid trees from individual loci showed considerable incongruity but little reticulation signal when support from more than one gene tree was required to infer reticulation. This was coupled with generally low support in the individual gene trees. Sequencing the duplicated gene copies in tetraploids showed clearer evidence of hybrid evolution, including multiple origins of one group of tetraploids included in the study.

Conclusions

A combination of cloning duplicate gene copies in allotetraploids and consensus network comparison of gene trees allowed a phylogenetic framework for reticulation in Polystachya to be built. There was little evidence for homoploid hybridization, but our knowledge of the origins and relationships of three groups of allotetraploids are greatly improved by this study. One group showed evidence of multiple long-distance dispersals to achieve a pantropical distribution; another showed no evidence of multiple origins or long-distance dispersal but had greater morphological variation, consistent with hybridization between more distantly related parents.     

Modified immunoslotblot assay to detect hemi and sulfur mustard DNA adducts

Sulfur mustard (SM) is an old chemical warfare agent causing blisters (vesicant). Skin toxicity is thought to be partly caused by SM induced DNA damage. SM and the hemi mustard 2-chloroethyl ethyl sulfide (CEES) are bi- and monofunctional DNA alkylating agents, respectively. Both chemicals react especially with N₇ guanine. The most abundant adducts are 7-hydroxyethylthioethylguanine for SM (61%) and 7-ethyl thioethylguanine for CEES. Thus, DNA alkylation should serve as a biomarker of SM exposure. A specific monoclonal antibody (2F8) was previously developed to detect SM and CEES adducts at N₇ position by means of immunoslotblot (ISB) technique (van der Schans et al. (2004) [16]). Nitrogen mustards (HN-1, HN-2, HN-3) are alkylating agents with structural similarities, which can form DNA adducts with N₇ guanine. The aim of the presented work was to modify the van der Schans protocol for use in a field laboratory and to test the cross reactivity of the 2F8 antibody against nitrogen mustards. Briefly, human keratinocytes were exposed to SM and CEES (0–300 μM, 60 min) or HN-1, HN-2, HN-3 (120 min). After exposure, cells were scraped and DNA was isolated and normalized. 1 μg DNA was transferred to a nitrocellulose membrane using a slotblot technique. After incubation with 2F8 antibody, the DNA adducts were visualized with chromogen staining (3,3′-diaminobenzidine (DAB), SeramunGrün). Blots were photographed and signal intensity was quantified. In general, DAB was superior to SeramunGrün stain. A staining was seen from 30 nM to 300 μM of SM or CEES, respectively. However, statistically significant DNA adducts were detected after CEES and SM exposure above 30 μM which is below the vesicant threshold. No signal was observed after HN-1, HN-2, HN-3 exposure. The total hands-on time to complete the assay was about 36 h. Further studies are necessary to validate SM or CEES exposure in blister roofs of exposed patients.     

SNAP-tag technology mediates site specific conjugation of antibody fragments with a photosensitizer and improves target specific phototoxicity in tumor cells

Cancer cells can be killed by photosensitizing agents that induce toxic effects when exposed to nonhazardous light, but this also causes significant damage to surrounding healthy cells. The specificity of photodynamic therapy can be increased by conjugating photosensitizing agents to antibodies and antibody fragments that bind specifically to tumor cell antigens. However, standard conjugation reactions produce heterogeneous products whose targeting specificity and spectroscopic properties can be compromised. In this study, we used an antibody fragment (scFv-425) that binds to the epidermal growth factor receptor (EGFR) as a model to investigate the use of SNAP-tag fusions as an improved conjugation strategy. The scFv-425-SNAP-tag fusion protein allowed the specific conjugation of a chlorin e6 photosensitizer modified with O(6)-benzylguanine, generating a homogeneous product that was delivered specifically to EGFR(+) cancer cells and resulted in significant, tumor cell-specific cytotoxicity. The impact of our results on the development of photodynamic therapy is discussed.     

Impacts of coral bleaching on pH and oxygen gradients across the coral concentration boundary layer: a microsensor study

Coral Reefs - Reef-building corals are surrounded by complex microenvironments (i.e. concentration boundary layers) that partially isolate them from the ambient seawater. Although the presence of...     

In vivo control of CpG and non-CpG DNA methylation by DNA methyltransferases

The enzymatic control of the setting and maintenance of symmetric and non-symmetric DNA methylation patterns in a particular genome context is not well understood. Here, we describe a comprehensive analysis of DNA methylation patterns generated by high resolution sequencing of hairpin-bisulfite amplicons of selected single copy genes and repetitive elements (LINE1, B1, IAP-LTR-retrotransposons, and major satellites). The analysis unambiguously identifies a substantial amount of regional incomplete methylation maintenance, i.e. hemimethylated CpG positions, with variant degrees among cell types. Moreover, non-CpG cytosine methylation is confined to ESCs and exclusively catalysed by Dnmt3a and Dnmt3b. This sequence position-, cell type-, and region-dependent non-CpG methylation is strongly linked to neighboring CpG methylation and requires the presence of Dnmt3L. The generation of a comprehensive data set of 146,000 CpG dyads was used to apply and develop parameter estimated hidden Markov models (HMM) to calculate the relative contribution of DNA methyltransferases (Dnmts) for de novo and maintenance DNA methylation. The comparative modelling included wild-type ESCs and mutant ESCs deficient for Dnmt1, Dnmt3a, Dnmt3b, or Dnmt3a/3b, respectively. The HMM analysis identifies a considerable de novo methylation activity for Dnmt1 at certain repetitive elements and single copy sequences. Dnmt3a and Dnmt3b contribute de novo function. However, both enzymes are also essential to maintain symmetrical CpG methylation at distinct repetitive and single copy sequences in ESCs.     

Annexin A8 regulates late endosome organization and function

Different classes of endosomes exhibit a characteristic intracellular steady-state distribution governed by interactions with the cytoskeleton. Late endosomes, organelles of the degradative lysosomal route, seem to require associated actin filaments for proper localization and function. We show here that the F-actin and phospholipid binding protein annexin A8 is associated specifically with late endosomes. Altering intracellular annexin A8 levels drastically affected the morphology and intracellular distribution of late endosomes. Trafficking through the degradative pathway was delayed in the absence of annexin A8, resulting in attenuated ligand-induced degradation of the epidermal growth factor receptor and prolonged epidermal growth factor-induced activation of mitogen-activated protein kinase. Depletion of annexin A8 reduced the association of late endosomal membranes with actin filaments. These results indicate that the defective cargo transport through the late endocytic pathway and the imbalanced signaling of activated receptors observed in the absence of annexin A8 results from the disturbed association of late endosomal membranes with the actin network, resulting in impaired actin-based late endosome motility.