Insights into Peroxisome Function from the Structure of PEX3 in Complex with a Soluble Fragment of PEX19

The human peroxins PEX3 and PEX19 play a central role in peroxisomal membrane biogenesis. The membrane-anchored PEX3 serves as the receptor for cytosolic PEX19, which in turn recognizes newly synthesized peroxisomal membrane proteins. After delivering these proteins to the peroxisomal membrane, PEX19 is recycled to the cytosol. The molecular mechanisms underlying these processes are not well understood. Here, we report the crystal structure of the cytosolic domain of PEX3 in complex with a PEX19-derived peptide. PEX3 adopts a novel fold that is best described as a large helical bundle. A hydrophobic groove at the membrane-distal end of PEX3 engages the PEX19 peptide with nanomolar affinity. Mutagenesis experiments identify phenylalanine 29 in PEX19 as critical for this interaction. Because key PEX3 residues involved in complex formation are highly conserved across species, the observed binding mechanism is of general biological relevance.     

White biotechnology for cellulose manufacturing—The HoLiR concept

A variety of approaches are available for generation of bacteria‐produced nanocellulose (BNC) in different forms. BNC production under static cultivation conditions usually results in fleeces or foils, characterized by a homogeneous, three‐dimensional network of nanofibers and a uniform surface. However, under static cultivation conditions in batch vessels, the widths and the lengths of the BNC sheets cultured are determined by the dimensions of the culture vessel. In this contribution, a novel, efficient process for a (semi‐)continuous cultivation of planar BNC fleeces and foils with a freely selectable length and an adjustable height is presented. By means of comprehensive investigations, the comparability of the BNC harvested to that gained from static cultivation under batch conditions is demonstrated. A first estimation of the production costs further shows that this type of processing allows for significant cost reductions compared to static cultivation of BNC in Erlenmeyer flasks. Biotechnol. Bioeng. 2010. 105: 740–747. © 2009 Wiley Periodicals, Inc.     

Immunological characteristics correlating with clinical response to immunotherapy in patients with advanced metastatic melanoma

Current treatment options for advanced metastatic melanoma are limited to experimental regimen that provide poor survival outcomes. Immunotherapy is a promising alternative and we recently reported a clinical trial in which 6 out of 19 patients enrolled had objective clinical responses to a fully autologous melanoma/dendritic cell vaccine. The mechanism of the vaccine is not well understood, but we hypothesized that general immunocompetence may be a determinant of clinical response. We therefore examined the immune status of an expanded series of 21 patients who displayed varying clinical responses to the melanoma/dendritic cell vaccine. Immunocompetence was assessed using in vitro assays of lymphocyte function: survival, proliferation and cytokine responses to mitogen stimulation as well as T-cell receptor zeta expression and lymphocyte subset analysis. Although lymphocytes from patients mostly performed comparably to age-matched and sex-matched controls, in some assays we identified significant differences between complete clinical responders and other patients, both before and following vaccination. Surprisingly, before vaccination, only lymphocytes from clinical responder patients showed impaired in vitro survival. Following vaccination, T lymphocyte survival improved and cells recovered their ability to produce the Th1-associated cytokines TNF and IFN-gamma in response to anti-CD3 stimulation in vitro. No increase in Th1 cytokine production was observed in lymphocytes from patients who experienced partial clinical responses or progressive disease. We conclude that, before vaccination, patients who go on to have complete responses have immune characteristics suggestive of high cell turnover and low Th1-associated cytokine production, and that these can be reversed with vaccination. These results have potential implications for future immunotherapeutic strategies.     

Will my protein crystallize? A sequence‐based predictor

We propose a machine-learning approach to sequence-based prediction of protein crystallizability in which we exploit subtle differences between proteins whose structures were solved by X-ray analysis [or by both X-ray and nuclear magnetic resonance (NMR) spectroscopy] and those proteins whose structures were solved by NMR spectroscopy alone. Because the NMR technique is usually applied on relatively small proteins, sequence length distributions of the X-ray and NMR datasets were adjusted to avoid predictions biased by protein size. As feature space for classification, we used frequencies of mono-, di-, and tripeptides represented by the original 20-letter amino acid alphabet as well as by several reduced alphabets in which amino acids were grouped by their physicochemical and structural properties. The classification algorithm was constructed as a two-layered structure in which the output of primary support vector machine classifiers operating on peptide frequencies was combined by a second-level Naive Bayes classifier. Due to the application of metamethods for cost sensitivity, our method is able to handle real datasets with unbalanced class representation. An overall prediction accuracy of 67% [65% on the positive (crystallizable) and 69% on the negative (noncrystallizable) class] was achieved in a 10-fold cross-validation experiment, indicating that the proposed algorithm may be a valuable tool for more efficient target selection in structural genomics. A Web server for protein crystallizability prediction called SECRET is available at http://webclu.bio.wzw.tum.de:8080/secret.     

Production and characterization of neutralizing monoclonal antibodies that recognize an epitope in domain 2 of Bacillus anthracis protective antigen

Antibodies against the protective antigen (PA) of Bacillus anthracis play a key role in response to infection by this important pathogen. The aim of this study was to produce and characterize monoclonal antibodies (mAbs) specific for PA and to identify novel neutralizing epitopes. Three murine mAbs with high specificity and nanomolar affinity for B. anthracis recombinant protective antigen (rPA) were produced and characterized. Western immunoblot analysis, coupled with epitope mapping using overlapping synthetic peptides, revealed that these mAbs recognize a linear epitope within domain 2 of rPA. Neutralization assays demonstrate that these mAbs effectively neutralize lethal toxin in vitro.     

Inefficient serotype knock down leads to stable coexistence of different surface antigens on the outer membrane in Paramecium tetraurelia

Expression of surface antigens is usually mutually exclusive, meaning that only one protein is present on the cell surface. With the RNAi feeding technology we induce serotype shifts in Paramecium tetraurelia which are demonstrated to be incomplete, meaning that the cells remain in a shifting state. The coexpression of "old" and "new" protein on the surface can be detected to be stable for more than 15 divisions over a 5-day feeding procedure, a time period different from that reported for temperature-induced shifts. A characteristic heterogenic distribution of the different surface antigens is demonstrated by double indirect-immunofluorescent-staining and we show antigen transport mechanisms related to the tips of cilia. Therefore, we discuss release mechanisms, potential sorting mechanisms for glycosylphosphatidylinositol-anchored proteins and the localizations of surface antigens, which are important for the reported classical immobilization reaction.     

Exchange of a single amino acid switches the substrate properties of RhoA and RhoD toward glucosylating and transglutaminating toxins

Rho GTPases are the preferred targets of various bacterial cytotoxins, including Clostridium difficile toxins A and B, Clostridium sordellii lethal toxin, the cytotoxic necrotizing factors (CNF1) from Escherichia coli, and the dermonecrotizing toxin (DNT) from Bordetella species. The toxins inactivate or activate specific sets of Rho GTPases by mono-O-glucosylation and deamidation/transglutamination, respectively. Here we studied the structural basis of the recognition of RhoA, which is modified by toxin B, CNF1, and DNT, in comparison with RhoD, which is solely a substrate for lethal toxin. We found that a single amino acid residue in RhoA and RhoD defines the substrate specificity for toxin B and lethal toxin. Change of serine 73 to phenylalanine in RhoA turned RhoA into a substrate for lethal toxin. Accordingly, change of the equivalently positioned phenylalanine 85 in RhoD with serine allowed glucosylation by toxin B. Comparable results were achieved with the Rho-activating and transglutaminating enzymes CNF1 and DNT. Here, amino acid glutamate 64 of RhoA and the equivalent aspartate 76 of RhoD define substrate specificity for CNF1 and DNT, respectively. These data indicate that single amino acid residues located in the switch II region of Rho proteins determine enzyme specificity for diverse bacterial toxins.     

The yeast Arr4p ATPase binds the chloride transporter Gef1p when copper is available in the cytosol

Cellular ion homeostasis involves communication between the cytosol and the luminal compartment of organelles. This is particularly critical for metal ions because of their toxic potential. We have identified the yeast homologue of the prokaryotic ArsA protein, the homodimeric ATPase Arr4p, as a protein that binds to the yeast intracellular CLC chloride-transport protein, Gef1p. We show that binding of Arr4p to the C terminus of Gef1p requires the presence of yeast cytosol and is sensitive to a highly specific copper chelator in vitro and in vivo. Copper alone can substitute for cytosol to support the interaction of Arr4p with the C terminus of Gef1p. The migration behavior of Arr4p in nonreducing gel electrophoresis correlates with cellular copper deficiency, repletion, or stress. Our homology model of Arr4p shows that the antimony (arsenic) metal binding site of ArsA is not conserved in Arr4p. The model suggests that a pair of cysteines, Cys285 and Cys288, is located in the interface of the Arr4p dimer. These residues are required for Arr4p homodimerization and for binding to the C terminus of Gef1p. Whereas both proteins are required for normal growth under iron-limiting conditions, they play opposite roles when copper and heat stress are combined in an alkaline environment. Under these conditions, deltagef1 cells grow much better than wild type yeast, whereas deltaarr4 cells are unable to grow. Comparison of the deltaarr4 with the deltaarr4deltagef1 strain suggests that Arr4p antagonizes the function of Gef1p.     

Soil seed banks near rubbing trees indicate dispersal of plant species into forests by wild boar

Current knowledge about processes that generate long-distance dispersal of plants is still limited despite its importance for persistence of populations and colonization of new potential habitats. Today wild large mammals are presumed to be important vectors for long-distance transport of diaspores within and between European temperate forest patches, and in particular wild boars recently came into focus. Here we use a specific habit of wild boar, i.e. wallowing in mud and subsequent rubbing against trees, to evaluate epizoochorous dispersal of vascular plant diaspores. We present soil seed bank data from 27 rubbing trees versus 27 control trees from seven forest areas in Germany. The mean number of viable seeds and the plant species number were higher in soil samples near rubbing trees compared with control trees. Ten of the 20 most frequent species were more frequent, and many species exclusively appeared in the soil samples near rubbing trees. The large number of plant species and seeds – more than 1000 per tree – in the soils near rubbing trees is difficult to explain unless the majority were dispersed by wild boar. Hooked and bristly diaspores, i.e. those adapted to epizoochory, were more frequent; however, many species with unspecialized diaspores occurred exclusively near rubbing trees. As opposed to plant species closely tied to forests species which occur both in forest and open vegetation and non-forest species were more frequent near rubbing trees compared with controls. These findings are consistent with previous studies on diaspore loads in the coats and hooves of shot wild boars. However, our method allows to identify the transport of diaspores from the open landscape into forest stands, where they might especially emerge after disturbance, and a clustered distribution of epizoochorically dispersed seeds. Moreover, accumulation of seeds of wetness indicators near rubbing trees demonstrates directed dispersal of plant species inhabiting wet places among remote wallows.