Identification and characterization of novel ERC‐55 interacting proteins: Evidence for the existence of several ERC‐55 splicing variants; including the cytosolic ERC‐55‐C

ERC‐55, encoded from RCN2, is localized in the ER and belongs to the CREC protein family. ERC‐55 is involved in various diseases and abnormal cell behavior, however, the function is not well defined and it has controversially been reported to interact with a cytosolic protein, the vitamin D receptor. We have used a number of proteomic techniques to further our functional understanding of ERC‐55. By affinity purification, we observed interaction with a large variety of proteins, including those secreted and localized outside of the secretory pathway, in the cytosol and also in various organelles. We confirm the existence of several ERC‐55 splicing variants including ERC‐55‐C localized in the cytosol in association with the cytoskeleton. Localization was verified by immunoelectron microscopy and sub‐cellular fractionation. Interaction of lactoferrin, S100P, calcyclin (S100A6), peroxiredoxin‐6, kininogen and lysozyme with ERC‐55 was further studied in vitro by SPR experiments. Interaction of S100P requires [Ca²⁺] of ～10^?7 M or greater, while calcyclin interaction requires [Ca²⁺] of >10^?5 M. Interaction with peroxiredoxin‐6 is independent of Ca²⁺. Co‐localization of lactoferrin, S100P and calcyclin with ERC‐55 in the perinuclear area was analyzed by fluorescence confocal microscopy. The functional variety of the interacting proteins indicates a broad spectrum of ERC‐55 activities such as immunity, redox homeostasis, cell cycle regulation and coagulation.     

A generic method for assignment of reliability scores applied to solvent accessibility predictions

Background  

Estimation of the reliability of specific real value predictions is nontrivial and the efficacy of this is often questionable. It is important to know if you can trust a given prediction and therefore the best methods associate a prediction with a reliability score or index. For discrete qualitative predictions, the reliability is conventionally estimated as the difference between output scores of selected classes. Such an approach is not feasible for methods that predict a biological feature as a single real value rather than a classification. As a solution to this challenge, we have implemented a method that predicts the relative surface accessibility of an amino acid and simultaneously predicts the reliability for each prediction, in the form of a Z-score.     

Ocular proteomics with emphasis on two-dimensional gel electrophoresis and mass spectrometry

The intention of this review is to provide an overview of current methodologies employed in the rapidly developing field of ocular proteomics with emphasis on sample preparation, two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) and mass spectrometry (MS). Appropriate sample preparation for the diverse range of cells and tissues of the eye is essential to ensure reliable results. Current methods of protein staining for 2D-PAGE, protein labelling for two-dimensional difference gel electrophoresis, gel-based expression analysis and protein identification by MS are summarised. The uses of gel-free MS-based strategies (MuDPIT, iTRAQ, ICAT and SILAC) are also discussed. Proteomic technologies promise to shed new light onto ocular disease processes that could lead to the discovery of strong novel biomarkers and therapeutic targets useful in many ophthalmic conditions.     

The role of cladocerans in tracking long-term change in shallow lake trophic status

Shallow lakes have been affected by a variety of human activities profoundly altering their ecological structure and function. Cladocerans have been used to track change resulting from a variety of drivers at a number of time scales. Aquatic macrophytes are well recognised as reflecting the ecological condition of a lake. Here, we compare the plant macrofossils with the sub-fossil cladoceran assemblages from 20 dated sediment cores. Co-correspondence analysis was used to determine the degree of commonality of change in community composition of the two biological groups through time. This analysis revealed very high levels of agreement in the nature and timing of change at all the sites examined with very high correlation coefficients between the axis 1 scores for macrofossils and cladocerans. Furthermore, at all sites a high proportion of the variance (min 20%, max 54%) in the macrofossil data was explained by the change in the cladoceran assemblage. Sub-fossil macrofossil and cladoceran assemblages, from at least from 1700 AD onwards, were examined in more detail at three sites: Ormesby Great Broad, Felbrigg Lake and Lake Søbygaard. There was very good accord in the main shifts of the cladoceran and macrofossil assemblages at all three sites. This may reflect the long-term shift in the principal focus of primary production from the benthic to the pelagic habitat. We suggest that the combination of their central position in the food-web and the presence of both pelagic and benthic taxa make cladocerans a strong candidate as the single best indicator of (palaeo) ecological condition related to changing trophic status and alteration in food-web structure in shallow lakes.     

Root-associated ectomycorrhizal fungi shared by various boreal forest seedlings naturally regenerating after a fire in interior alaska and correlation of different fungi with host growth responses

The role of common mycorrhizal networks (CMNs) in postfire boreal forest successional trajectories is unknown. We investigated this issue by sampling a 50-m by 40-m area of naturally regenerating black spruce (Picea mariana), trembling aspen (Populus tremuloides), and paper birch (Betula papyrifera) seedlings at various distances from alder (Alnus viridis subsp. crispa), a nitrogen-fixing shrub, 5 years after wildfire in an Alaskan interior boreal forest. Shoot biomasses and stem diameters of 4-year-old seedlings were recorded, and the fungal community associated with ectomycorrhizal (ECM) root tips from each seedling was profiled using molecular techniques. We found distinct assemblages of fungi associated with alder compared with those associated with the other tree species, making the formation of CMNs between them unlikely. However, among the spruce, aspen, and birch seedlings, there were many shared fungi (including members of the Pezoloma ericae [Hymenoscyphus ericae] species aggregate, Thelephora terrestris, and Russula spp.), raising the possibility that these regenerating seedlings may form interspecies CMNs. Distance between samples did not influence how similar ECM root tip-associated fungal communities were, and of the fungal groups identified, only one of them was more likely to be shared between seedlings that were closer together, suggesting that the majority of fungi surveyed did not have a clumped distribution across the small scale of this study. The presence of some fungal ribotypes was associated with larger or smaller seedlings, suggesting that these fungi may play a role in the promotion or inhibition of seedling growth. The fungal ribotypes associated with larger seedlings were different between spruce, aspen, and birch, suggesting differential impacts of some host-fungus combinations. One may speculate that wildfire-induced shifts in a given soil fungal community could result in variation in the growth response of different plant species after fire and a shift in regenerating vegetation.     

Substrate specificity of the bovine serum amine oxidase and in situ characterisation of aminoaldehydes by NMR spectroscopy

The oxidation of spermidine or homospermidine with bovine serum amine oxidase (BSAO) was monitored in situ, using proton nuclear magnetic resonance spectroscopy in water with 10% D(2)O. NMR assignments were performed by spin decoupling and COSY spectra or by comparison with data from synthetic aminoaldehydes. The results represent the first in situ characterisation of the highly reactive aminoaldehydes and showed oxidation at the N(1) amino group of spermidine and homospermidine. Comparison of homospermidine with a variety of substrates revealed that among straight chain di- and polyamines both an aminopropyl group and two primary amino groups separated by seven (norspermidine) or eight (spermidine) carbon atoms were required for optimal substrate ability. However, highest activity was seen with the substrate N-(4-aminobutyl)hexahydropyrimidine, showing that the substrate channel of BSAO has a dual substrate preference, with moderately bulky substituents at the distal end of a diamine contributing equally well as an alkyl amino group. Cytotoxic investigations of a variety of substrates for BSAO, confirmed previous results, that cytotoxicity is primarily linked to polyamines encompassing the aminopropyl moiety. No acrolein was observed at any time during the oxidation showing that it reacts very fast with available amino groups forming a variety of derivatives.     

FLS2-BAK1 Extracellular Domain Interaction Sites Required for Defense Signaling Activation

Signaling initiation by receptor-like kinases (RLKs) at the plasma membrane of plant cells often requires regulatory leucine-rich repeat (LRR) RLK proteins such as SERK or BIR proteins. The present work examined how the microbe-associated molecular pattern (MAMP) receptor FLS2 builds signaling complexes with BAK1 (SERK3). We first, using in vivo methods that validate separate findings by others, demonstrated that flg22 (flagellin epitope) ligand-initiated FLS2-BAK1 extracellular domain interactions can proceed independent of intracellular domain interactions. We then explored a candidate SERK protein interaction site in the extracellular domains (ectodomains; ECDs) of the significantly different receptors FLS2, EFR (MAMP receptors), PEPR1 (damage-associated molecular pattern (DAMP) receptor), and BRI1 (hormone receptor). Repeat conservation mapping revealed a cluster of conserved solvent-exposed residues near the C-terminus of models of the folded LRR domains. However, site-directed mutagenesis of this conserved site in FLS2 did not impair FLS2-BAK1 ECD interactions, and mutations in the analogous site of EFR caused receptor maturation defects. Hence this conserved LRR C-terminal region apparently has functions other than mediating interactions with BAK1. In vivo tests of the subsequently published FLS2-flg22-BAK1 ECD co-crystal structure were then performed to functionally evaluate some of the unexpected configurations predicted by that crystal structure. In support of the crystal structure data, FLS2-BAK1 ECD interactions were no longer detected in in vivo co-immunoprecipitation experiments after site-directed mutagenesis of the FLS2 BAK1-interaction residues S554, Q530, Q627 or N674. In contrast, in vivo FLS2-mediated signaling persisted and was only minimally reduced, suggesting residual FLS2-BAK1 interaction and the limited sensitivity of co-immunoprecipitation data relative to in vivo assays for signaling outputs. However, Arabidopsis plants expressing FLS2 with the Q530A+Q627A double mutation were impaired both in detectable interaction with BAK1 and in FLS2-mediated responses, lending overall support to current models of FLS2 structure and function.     

Similarity between contemporary vegetation and plant remains in the surface sediment in Mediterranean lakes

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