Endoplasmic Reticulum-Dependent Redox Reactions Control Endoplasmic Reticulum-Associated Degradation and Pathogen Entry

Abstract Significance: Protein misfolding within the endoplasmic reticulum (ER) is managed by an ER quality control system that retro-translocates aberrant proteins into the cytosol for proteasomal destruction. This process, known as ER-associated degradation, utilizes the action of ER redox enzymes to accommodate the disulfide-bonded nature of misfolded proteins. Strikingly, various pathogenic viruses and toxins co-opt these redox components to reach the cytosol during entry. These redox factors thus regulate critical cellular homeostasis and host?pathogen interactions. Recent Advances: Recent studies identify specific members of the protein disulfide isomerase (PDI) family, which use their chaperone and catalytic activities, in engaging both misfolded ER proteins and pathogens. Critical Issues: The precise molecular mechanism by which a dedicated PDI family member disrupts the disulfide bonds in the misfolded ER proteins and pathogens, as well as how they act to unfold these substrates to promote their ER-to-cytosol membrane transport, remain poorly characterized. Future Directions: How PDI family members distinguish folded versus misfolded ER substrates remains enigmatic. What physical characteristics surrounding a substrate's disulfide bond instruct PDI that it is mispaired or native? For the pathogens, as their disulfide bonds normally serve a critical role in providing physical support, what conformational changes experienced in the host enable their disulfide bonds to be disrupted? A combination of more rigorous biochemical and high-resolution structural studies should begin to address these questions. Antioxid. Redox Signal. 16, 809?818.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/98492/1/ars%2E2011%2E4425.pd

A Therapeutic Chemical Chaperone Inhibits Cholera Intoxication and Unfolding/Translocation of the Cholera Toxin A1 Subunit

Cholera toxin (CT) travels as an intact AB5 protein toxin from the cell surface to the endoplasmic reticulum (ER) of an intoxicated cell. In the ER, the catalytic A1 subunit dissociates from the rest of the toxin. Translocation of CTA1 from the ER to the cytosol is then facilitated by the quality control mechanism of ER-associated degradation (ERAD). Thermal instability in the isolated CTA1 subunit generates an unfolded toxin conformation that acts as the trigger for ERAD-mediated translocation to the cytosol. In this work, we show by circular dichroism and fluorescence spectroscopy that exposure to 4-phenylbutyric acid (PBA) inhibited the thermal unfolding of CTA1. This, in turn, blocked the ER-to-cytosol export of CTA1 and productive intoxication of either cultured cells or rat ileal loops. In cell culture studies PBA did not affect CT trafficking to the ER, CTA1 dissociation from the holotoxin, or functioning of the ERAD system. PBA is currently used as a therapeutic agent to treat urea cycle disorders. Our data suggest PBA could also be used in a new application to prevent or possibly treat cholera

Core charge distribution and self assembly of columnar phases: the case of triphenylenes and azatriphenylenes

<p>Abstract</p> <p>Background</p> <p>The relation betweeen the structure of discotic molecules and columnar properties, a crucial point for the realization of new advanced materials, is still largely unknown. A paradigmatic case is that hexa-alkyl-thio substituted triphenylenes present mesogenic behavior while the corresponding azatriphenylenes, similar in shape and chemical structure, but with a different core charge distribution, do not form any liquid crystalline mesophase. This study is aimed at investigating, with the help of computer simulations techniques, the effects on phase behaviour of changes of the charge distribution in the discotic core.</p> <p>Results</p> <p>We described the shape and the pair, dispersive and electrostatic, interactions of hexa alkyl triphenylenes by uniaxial Gay-Berne discs with embedded point charges. Gay-Berne parameters were deduced by fitting the dispersive energies obtained from an atomistic molecular dynamics simulation of a small sample of hexa-octyl-thio triphenylene molecules in columnar phase, while a genetic algorithm was used to get a minimal set of point charges that properly reproduces the ab anitio electrostatic potential. We performed Monte Carlo simulations of three molecular models: the pure Gay-Berne disc, used as a reference, the Gay-Berne disc with hexa-thio triphenylene point charges, the Gay-Berne disc with hexa-thio azatriphenylene point charges. The phase diagram of the pure model evidences a rich polymorphism, with isotropic, columnar and crystalline phases at low pressure, and the appearance of nematic phase at higher pressure.</p> <p>Conclusion</p> <p>We found that the intermolecular electrostatic potential among the cores is fundamental in sta-bilizing/destabilizing columnar phases; in particular the triphenylene charge distribution stabilizes the columnar structure, while the azatriphenylene distribution suppresses its formation in favor of the nematic phase. We believe the present model could be successfully employed as the basis for coarse-grained level simulations of a wider class of triphenylene derivatives.</p

An EGF-like Protein Forms a Complex with PfRh5 and Is Required for Invasion of Human Erythrocytes by Plasmodium falciparum

Invasion of erythrocytes by Plasmodium falciparum involves a complex cascade of protein-protein interactions between parasite ligands and host receptors. The reticulocyte binding-like homologue (PfRh) protein family is involved in binding to and initiating entry of the invasive merozoite into erythrocytes. An important member of this family is PfRh5. Using ion-exchange chromatography, immunoprecipitation and mass spectroscopy, we have identified a novel cysteine-rich protein we have called P. falciparum Rh5 interacting protein (PfRipr) (PFC1045c), which forms a complex with PfRh5 in merozoites. Mature PfRipr has a molecular weight of 123 kDa with 10 epidermal growth factor-like domains and 87 cysteine residues distributed along the protein. In mature schizont stages this protein is processed into two polypeptides that associate and form a complex with PfRh5. The PfRipr protein localises to the apical end of the merozoites in micronemes whilst PfRh5 is contained within rhoptries and both are released during invasion when they form a complex that is shed into the culture supernatant. Antibodies to PfRipr1 potently inhibit merozoite attachment and invasion into human red blood cells consistent with this complex playing an essential role in this process

Evaluation of allelic forms of the erythrocyte binding antigen 175 (EBA-175) in Plasmodium falciparum field isolates from Brazilian endemic area

<p>Abstract</p> <p>Background</p> <p>The <it>Plasmodium falciparum </it>Erythrocyte Binding Antigen-175 (EBA-175) is an antigen considered to be one of the leading malaria vaccine candidates. EBA-175 mediates sialic acid-dependent binding to glycophorin A on the erythrocytes playing a crucial role during invasion of the <it>P. falciparum </it>in the host cell. Dimorphic allele segments, termed C-fragment and F-fragment, have been found in high endemicity malaria areas and associations between the dimorphism and severe malaria have been described. In this study, the genetic dimorphism of EBA-175 was evaluated in <it>P. falciparum </it>field isolates from Brazilian malaria endemic area.</p> <p>Methods</p> <p>The study was carried out in rural villages situated near Porto Velho, Rondonia State in the Brazilian Amazon in three time points between 1993 and 2008. The allelic dimorphism of the EBA-175 was analysed by Nested PCR.</p> <p>Results</p> <p>The classical allelic dimorphism of the EBA-175 was identified in the studied area. Overall, C-fragment was amplified in a higher frequency than F-fragment. The same was observed in the three time points where C-fragment was observed in a higher frequency than F-fragment. Single infections (one fragment amplified) were more frequent than mixed infection (two fragments amplified).</p> <p>Conclusions</p> <p>These findings confirm the dimorphism of EBA175, since only the two types of fragments were amplified, C-fragment and F-fragment. Also, the results show the remarkable predominance of CAMP allele in the studied area. The comparative analysis in three time points indicates that the allelic dimorphism of the EBA-175 is stable over time.</p

Leishmania major Survival in Selective Phlebotomus papatasi Sand Fly Vector Requires a Specific SCG-Encoded Lipophosphoglycan Galactosylation Pattern

Phlebotomine sand flies that transmit the protozoan parasite Leishmania differ greatly in their ability to support different parasite species or strains in the laboratory: while some show considerable selectivity, others are more permissive. In “selective” sand flies, Leishmania binding and survival in the fly midgut typically depends upon the abundant promastigote surface adhesin lipophosphoglycan (LPG), which exhibits species- and strain-specific modifications of the dominant phosphoglycan (PG) repeat units. For the “selective” fly Phlebotomus papatasi PpapJ, side chain galactosyl-modifications (scGal) of PG repeats play key roles in parasite binding. We probed the specificity and properties of this scGal-LPG PAMP (Pathogen Associated Molecular Pattern) through studies of natural isolates exhibiting a wide range of galactosylation patterns, and of a panel of isogenic L. major engineered to express similar scGal-LPG diversity by transfection of SCG-encoded β1,3-galactosyltransferases with different activities. Surprisingly, both ‘poly-scGal’ and ‘null-scGal’ lines survived poorly relative to PpapJ-sympatric L. major FV1 and other ‘mono-scGal’ lines. However, survival of all lines was equivalent in P. duboscqi, which naturally transmit L. major strains bearing ‘null-scGal’-LPG PAMPs. We then asked whether scGal-LPG-mediated interactions were sufficient for PpapJ midgut survival by engineering Leishmania donovani, which normally express unsubstituted LPG, to express a ‘PpapJ-optimal’ scGal-LPG PAMP. Unexpectedly, these “L. major FV1-cloaked” L. donovani-SCG lines remained unable to survive within PpapJ flies. These studies establish that midgut survival of L. major in PpapJ flies is exquisitely sensitive to the scGal-LPG PAMP, requiring a specific ‘mono-scGal’ pattern. However, failure of ‘mono-scGal’ L. donovani-SCG lines to survive in selective PpapJ flies suggests a requirement for an additional, as yet unidentified L. major-specific parasite factor(s). The interplay of the LPG PAMP and additional factor(s) with sand fly midgut receptors may determine whether a given sand fly host is “selective” or “permissive”, with important consequences to both disease transmission and the natural co-evolution of sand flies and Leishmania

Novel Serial Positive Enrichment Technology Enables Clinical Multiparameter Cell Sorting

A general obstacle for clinical cell preparations is limited purity, which causes variability in the quality and potency of cell products and might be responsible for negative side effects due to unwanted contaminants. Highly pure populations can be obtained best using positive selection techniques. However, in many cases target cell populations need to be segregated from other cells by combinations of multiple markers, which is still difficult to achieve – especially for clinical cell products. Therefore, we have generated low-affinity antibody-derived Fab-fragments, which stain like parental antibodies when multimerized via Strep-tag and Strep-Tactin, but can subsequently be removed entirely from the target cell population. Such reagents can be generated for virtually any antigen and can be used for sequential positive enrichment steps via paramagnetic beads. First protocols for multiparameter enrichment of two clinically relevant cell populations, CD4high/CD25high/CD45RAhigh ‘regulatory T cells’ and CD8high/CD62Lhigh/CD45RAneg ‘central memory T cells’, have been established to determine quality and efficacy parameters of this novel technology, which should have broad applicability for clinical cell sorting as well as basic research

Improvement in the Accuracy of Back Trajectories Using WRF to Identify Pollen Sources in Southern Iberian Peninsula

Airborne pollen transport at micro-, meso-gamma and meso-beta scales must be studied by atmospheric models, having special relevance in complex terrain. In these cases, the accuracy of these models is mainly determined by the spatial resolution of the underlying meteorological dataset. This work examines how meteorological datasets determine the results obtained from atmospheric transport models used to describe pollen transport in the atmosphere. We investigate the effect of the spatial resolution when computing backward trajectories with the HYSPLIT model. We have used meteorological datasets from the WRF model with 27, 9 and 3 km resolutions and from the GDAS files with 1 ° resolution. This work allows characterizing atmospheric transport of Olea pollen in a region with complex flows. The results show that the complex terrain affects the trajectories and this effect varies with the different meteorological datasets. Overall, the change from GDAS to WRF-ARW inputs improves the analyses with the HYSPLIT model, thereby increasing the understanding the pollen episode. The results indicate that a spatial resolution of at least 9 km is needed to simulate atmospheric flows that are considerable affected by the relief of the landscape. The results suggest that the appropriate meteorological files should be considered when atmospheric models are used to characterize the atmospheric transport of pollen on micro-, meso-gamma and meso-beta scales. Furthermore, at these scales, the results are believed to be generally applicable for related areas such as the description of atmospheric transport of radionuclides or in the definition of nuclear-radioactivity emergency preparedness

Evidence of niche partitioning under ontogenetic influences among three morphologically similar siluriformes in small subtropical streams

Ontogenetic influences in patterns of niche breadth and feeding overlap were investigated in three species of Siluriformes (Heptapterus sp., Rhamdia quelen and Trichomycterus poikilos) aiming at understanding the species coexistence. Samplings were conducted bimonthly by electrofishing technique from June/2012 to June/2013 in ten streams of the northwestern state of Rio Grande do Sul, Brazil. The stomach contents of 1,948 individuals were analyzed by volumetric method, with 59 food items identified. In general Heptapterus sp. consumed a high proportion of Aegla sp., terrestrial plant remains and Megaloptera; R. quelen consumed fish, and Oligochaeta, followed by Aegla sp.; while the diet of T. poikilos was based on Simuliidae, Ephemeroptera and Trichoptera. Specie segregation was observed in the NMDS. Through PERMANOVA analysis feeding differences among species, and between a combination of species plus size classes were observed. IndVal showed which items were indicators of these differences. Niche breadth values were high for all species. The niche breadth values were low only for the larger size of R. quelen and Heptapterus sp. while T. poikilos values were more similar. Overall the species were a low feeding overlap values. The higher frequency of high feeding overlap was observed for interaction between Heptapterus sp. and T. poikilos. The null model confirmed the niche partitioning between the species. The higher frequency of high and intermediate feeding overlap values were reported to smaller size classes. The null model showed resource sharing between the species/size class. Therefore, overall species showed a resource partitioning because of the use of occasional items. However, these species share resources mainly in the early ontogenetic stages until the emphasized change of morphological characteristics leading to trophic niche expansion and the apparent segregation observed