Interacting regions of CD81 and two of its partners, EWI-2 and EWI-2wint, and their effect on hepatitis C virus infection

CD81 is a tetraspanin protein that is involved in several essential cellular functions, as well as in the hepatitis C virus (HCV) infection. CD81 interacts with a high stoichiometry with its partner proteins EWI-2, EWI-2wint, and EWI-F. These latter proteins modify the functions of CD81 and can thereby potentially inhibit infection or modulate cell migration. Here, we characterized the cleavage of EWI-2 leading to the production of EWI-2wint, which has been shown to inhibit HCV infection. We determined the regions of EWI-2/EWI-2wint and CD81 that are important for their interaction and their functionality. More precisely, we identified a glycine zipper motif in the transmembrane domain of EWI-2/EWI-2wint that is essential for the interaction with CD81. In addition, we found that palmitoylation on two juxtamembranous cysteines in the cytosolic tail of EWI-2/EWI-2wint is required for their interaction with CD81 as well as with CD9, another tetraspanin. Thus, we have shown that palmitoylation of a tetraspanin partner protein can influence the interaction with a tetraspanin. We therefore propose that palmitoylation not only of tetraspanins, but also of their partner proteins is important in regulating the composition of complexes in tetraspanin networks. Finally, we identified the regions in CD81 that are necessary for its functionality in HCV entry and we demonstrated that EWI-2wint needs to interact with CD81 to exert its inhibitory effect on HCV infection.     

Spatial regulation of Cdc55-PP2A by Zds1/Zds2 controls mitotic entry and mitotic exit in budding yeast

Budding yeast CDC55 encodes a regulatory B subunit of the PP2A (protein phosphatase 2A), which plays important roles in mitotic entry and mitotic exit. The spatial and temporal regulation of PP2A is poorly understood, although recent studies demonstrated that the conserved proteins Zds1 and Zds2 stoichiometrically bind to Cdc55-PP2A and regulate it in a complex manner. Zds1/Zds2 promote Cdc55-PP2A function for mitotic entry, whereas Zds1/Zds2 inhibit Cdc55-PP2A function during mitotic exit. In this paper, we propose that Zds1/Zds2 primarily control Cdc55 localization. Cortical and cytoplasmic localization of Cdc55 requires Zds1/Zds2, and Cdc55 accumulates in the nucleus in the absence of Zds1/Zds2. By genetically manipulating the nucleocytoplasmic distribution of Cdc55, we showed that Cdc55 promotes mitotic entry when in the cytoplasm. On the other hand, nuclear Cdc55 prevents mitotic exit. Our analysis defines the long-sought molecular function for the zillion different screens family proteins and reveals the importance of the regulation of PP2A localization for proper mitotic progression.     

Cell lines, Md108 and Md66, from the hemocytes of Malacosoma disstria (Lepidoptera) display aspects of plasma-free innate non-self activities

The innate non-self response systems of the deciduous tree pest, the forest tent caterpillar, Malacosoma disstria has been documented by us in terms of in vitro and in vivo reactions towards the Gram-positive nonpathogenic bacterium, Bacillus subtilis and Gram-negative pathogenic microbe, Xenorhabdus nematophila and their respective surface antigens, lipopoteichoic acids (LTA) and lipopolysaccharides (LPS). These studies, often conducted in whole and diluted hemolymph, preclude examination of plasma-free cellular (hemocyte) responses. Plasma-free hemocytes as primary cultures are difficult to obtain. The floating cell line Md66 and attached cell line Md108 from M. disstria hemocytes were examined as a model for plasma-free M. disstria hemocyte non-self responses. Herein, it was established that although both lines differed from each other and from the primary hemocyte cultures of M. disstria in growth parameters, cell composition and sizes both cell lines displayed granular cell-like (GL) cells and plasmatocyte-like (PL) cells according to morphological criteria and to some extent antigenic similarities based on labeling with anti-Chrysodeixis includens hemocyte monoclonal antibodies. Hemocyte-specific neuroglian-like protein was detected on cells of both cell lines and in the primary hemocyte cultures albeit with staining patterns differing according to culture and cell types, confluency levels and cell–cell adhesion. Both cell lines bound B. subtilis and X. nematophila, the reaction extent varying with the cell line and its cell types. LPS damaged both cell types in the two cell lines whereas LTA enhanced the adhesion of Md66 GL cells to flask surfaces followed by PL cell adhesion. PL cells of both lines, like the primary cultures, phagocytosed FITC-labeled B. subtilis; only Md108 GL cells phagocytosed B. subtilis. In either case phagocytosis was always less in frequency and intensity than the primary cultures. Proteins released from the cell lines differed in pattern and magnitude but contained bacterial binding proteins that enhanced differential bacterial adhesion to both cell types in both cell lines: the GL cells both cultures, and those of granular cells in primary cultures, were more involved than the primary plasmatocytes and PL cells. Only Md66 cells possessed lysozyme and both cell types of both lines contained phenoloxidase. Neither enzyme type was released during early phase reaction with the bacteria. LPS inhibited phenoloxidase activity. The similarities and differences between the lines and primary cultures make Md66 and Md108 useful for the systematic examination of plasma-free cellular non-self reactions.     

Pollination and Reproductive Biology of Rauvolfia grandiflora (Apocynaceae): Secondary Pollen Presentation, Herkogamy and Self-Incompatibility

Abstract: The pollination and reproductive biology of Rauvolfia grandiflora were studied in natural populations in the forest of the Recife Botanical Garden. R. grandiflora is a shrubby species (2 to 5 m), the flowers are hermaphrodite and salverform. The corolla tube is white and the five free lobes of the corolla are flushed with violet. The five stamens are attached to the corolla tube; the anthers are introrse and form a cone situated immediately above the stigmatic head. There is a space between the anthers and the stigmatic head, where the pollen is deposited. The stigmatic head has three functional regions: a) an upper sterile region; b) a median region that produces a sticky substance and c) a lower receptive region, which is located beneath a basal collar. The secondary pollen presentation and the herkogamy mechanisms are discussed. Sugar concentration in nectar was, on average, 31.7%. The pollen viability was 97.4%. R. grandiflora is melittophilous and in the study area was found to be pollinated by only one species of long-tongued bee, Exaerete smaragdina. R. grandiflora was found to be self-incompatible, and the percentage of fruit set under natural conditions was low.     

The budding yeast PP2ACdc55 protein phosphatase prevents the onset of anaphase in response to morphogenetic defects

Faithful chromosome transmission requires establishment of sister chromatid cohesion during S phase, followed by its removal at anaphase onset. Sister chromatids are tethered together by cohesin, which is displaced from chromosomes through cleavage of its Mcd1 subunit by the separase protease. Separase is in turn inhibited, up to this moment, by securin. Budding yeast cells respond to morphogenetic defects by a transient arrest in G2 with high securin levels and unseparated chromatids. We show that neither securin elimination nor forced cohesin cleavage is sufficient for anaphase in these conditions, suggesting that other factors contribute to cohesion maintainance in G2. We find that the protein phosphatase PP2A bound to its regulatory subunit Cdc55 plays a key role in this process, uncovering a new function for PP2A(Cdc55) in controlling a noncanonical pathway of chromatid cohesion removal.     

The stress protein ERp57/GRP58 binds specific DNA sequences in HeLa cells

The protein ERp57/GRP58 is a member of the protein disulfide isomerase family and is also a glucose-regulated protein, which, together with the other GRPs, is induced by a variety of cellular stress conditions. ERp57/GRP58 is mainly located in the endoplasmic reticulum (ER), but has also been found in the cytoplasm and in the nucleus, where it can bind DNA. In order to identify a possible correlation between the stress-response and the nuclear location of ERp57/GRP58, its binding sites on DNA in HeLa cells have been searched by chromatin immunoprecipitation and cloning of the immunoprecipitated DNA fragments. Following sequencing of the cloned fragments, 10 DNA sequences have been securely identified as in vivo targets of ERp57/GRP58. Nine of them are present in the non-coding regions of identified genes, and seven of these in introns. The features of some of these DNA sequences, that is, DNase hypersensitivity, proximity of MAR regions, and homology to the non-coding regions of orthologue genes of mouse or rat, are compatible with a gene expression regulatory function. Considering the nature of the genes concerned, two of which code for DNA repair proteins, we would suggest that at least part of the mechanism of action of ERp57/GRP58 takes place through the regulation of these, and possibly other still unidentified, stress-response genes.     

Immunomodulatory mediators from pollen enhance the migratory capacity of dendritic cells and license them for Th2 attraction

The immune response of atopic individuals against allergens is characterized by increased levels of Th2 cytokines and chemokines. However, the way in which the cytokine/chemokine profile is matched to the type of invading allergen, and why these profiles sometimes derail and lead to disease, is not well understood. We recently demonstrated that pollen modulates dendritic cell (DC) function in a way that results in an enhanced capacity to initiate Th2 responses in vitro. Here, we examined the effects of aqueous birch pollen extracts (Bet.-APE) on chemokine receptor expression and chemokine production by human monocyte-derived DCs. Bet.-APE strongly induced expression and function of CXCR4 and reduced CCR1 and CCR5 expression on immature DCs. In addition, DC treatment with Bet.-APE significantly reduced LPS-induced production of CXCL10/IP-10, CCL5/RANTES; induced CCL22/macrophage-derived chemokine; and did not significantly change release of CCL17/thymus and activation-regulated chemokine. At a functional level, Bet.-APE increased the capacity of LPS-stimulated DCs to attract Th2 cells, whereas the capacity to recruit Th1 cells was reduced. Bet.-APE significantly and dose-dependently enhanced intracellular cAMP, suggesting that water-soluble factors from pollen grains bind a G(alphas)-protein-coupled receptor. E(1)-Phytoprostanes were identified to be one player in the Th2-polarizing potential of aqueous pollen extracts. In summary, our results demonstrate that pollen itself releases regulatory mediators which generate a Th2-promoting micromilieu with preferential recruitment of Th2 cells to the site of pollen exposure.     

X-ray crystal structures of human immunodeficiency virus type 1 protease mutants complexed with atazanavir

Atazanavir, which is marketed as REYATAZ, is the first human immunodeficiency virus type 1 (HIV-1) protease inhibitor approved for once-daily administration. As previously reported, atazanavir offers improved inhibitory profiles against several common variants of HIV-1 protease over those of the other peptidomimetic inhibitors currently on the market. This work describes the X-ray crystal structures of complexes of atazanavir with two HIV-1 protease variants, namely, (i) an enzyme optimized for resistance to autolysis and oxidation, referred to as the cleavage-resistant mutant (CRM); and (ii) the M46I/V82F/I84V/L90M mutant of the CRM enzyme, which is resistant to all approved HIV-1 protease inhibitors, referred to as the inhibitor-resistant mutant. In these two complexes, atazanavir adopts distinct bound conformations in response to the V82F substitution, which may explain why this substitution, at least in isolation, has yet to be selected in vitro or in the clinic. Because of its nearly symmetrical chemical structure, atazanavir is able to make several analogous contacts with each monomer of the biological dimer.