Effector granules in human T lymphocytes: the luminal proteome of secretory lysosomes from human T cells

Background

The incidence of cancer in patients with neurological diseases, who have been treated with LiCl, is below average. LiCl is a well-established inhibitor of Glycogen synthase kinase-3, a kinase that controls several cellular processes, among which is the degradation of the tumour suppressor protein p53. We therefore wondered whether LiCl induces p53-dependent cell death in cancer cell lines and experimental tumours.

Results

Here we show that LiCl induces apoptosis of tumour cells both in vitro and in vivo. Cell death was accompanied by cleavage of PARP and Caspases-3, -8 and -10. LiCl-induced cell death was not dependent on p53, but was augmented by its presence. Treatment of tumour cells with LiCl strongly increased TNF-α and FasL expression. Inhibition of TNF-α induction using siRNA or inhibition of FasL binding to its receptor by the Nok-1 antibody potently reduced LiCl-dependent cleavage of Caspase-3 and increased cell survival. Treatment of xenografted rats with LiCl strongly reduced tumour growth.

Conclusions

Induction of cell death by LiCl supports the notion that GSK-3 may represent a promising target for cancer therapy. LiCl-induced cell death is largely independent of p53 and mediated by the release of TNF-α and FasL. Key words: LiCl, TNF-α, FasL, apoptosis, GSK-3, FasL     

Macin family of antimicrobial proteins combines antimicrobial and nerve repair activities

The tertiary structures of theromacin and neuromacin confirmed the macin protein family as a self-contained family of antimicrobial proteins within the superfamily of scorpion toxin-like proteins. The macins, which also comprise hydramacin-1, are antimicrobially active against Gram-positive and Gram-negative bacteria. Despite high sequence identity, the three proteins showed distinct differences with respect to their biological activity. Neuromacin exhibited a significantly stronger capacity to permeabilize the cytoplasmic membrane of Bacillus megaterium than theromacin and hydramacin-1. Accordingly, it is the only macin that displays pore-forming activity and that was potently active against Staphylococcus aureus. Moreover, neuromacin and hydramacin-1 led to an aggregation of bacterial cells that was not observed with theromacin. Analysis of the molecular surface properties of macins allowed confirmation of the barnacle model as the mechanistic model for the aggregation effect. Besides being antimicrobially active, neuromacin and theromacin, in contrast to hydramacin-1, were able to enhance the repair of leech nerves ex vivo. Notably, all three macins enhanced the viability of murine neuroblastoma cells, extending their functional characteristics. As neuromacin appears to be both a functional and structural chimera of hydramacin-1 and theromacin, the putative structural correlate responsible for the nerve repair capacity in leech was located to a cluster of six amino acid residues using the sequence similarity of surface-exposed regions.     

Self-assembly of the isolated KCNQ2 subunit interaction domain

Mutations in the KCNQ2 gene cause myokymia and neonatal epilepsy, indicating that this K(+) channel regulates the excitability of lower motoneurons and CNS neurons. Little is known about the parameters that direct the assembly of this multimeric molecule and other KCNQ subunits. Here, we show that the carboxy-terminal subunit interaction domain of KCNQ2 autonomously folds and assembles into tetramers. This domain contains a bipartite coiled-coil motif. Whereas structural integrity of the second coiled-coil motif is crucial for tetramer formation, that of the first motif is less important. These data suggest a crucial role of coiled-coil motifs in tetrameric KCNQ channel assembly.     

2‐D DIGE analysis of the proteome of extracts from peanut variants reveals striking differences in major allergen contents

Over the last decade, an increasing prevalence of peanut allergies was observed worldwide. Peanuts are meanwhile categorized among the most dangerous food allergens. This is particularly relevant since peanut‐derived ingredients are widely used in industrial food production. To minimize the problem of hidden food allergens causing severe anaphylactic reactions, pre‐packaged food containing peanut components needs to be classified according to European ruling since 2005. Food companies search for strategies to reduce the allergenicity of peanut‐derived food additives either by genetically altering the allergen content or by identifying peanut varieties with low levels of major allergens. In our study, we focused on peanut extracts from Indonesia that apparently contain lower levels of the major Arachis hypogaea allergen 1 (Ara h 1). Basic extracts of Virginia‐type and Indonesian peanuts were compared by 1‐ and 2‐DE. We identified more than hundred individual components in these extracts by MS and provide a high‐resolution allergen map that also includes so far unknown fragments of major peanut allergens. The reduced level of Ara h 1 associated with a significantly lower abundance of the most potent peanut allergen Ara h 2 in various Indonesian peanuts was also confirmed by Western blotting with monoclonal antibodies and sera of allergic patients.     

An efficient fluorimetric method to measure the viability of intraerythrocytic Plasmodium falciparum

A range of various assays to measure chemosusceptibility of Plasmodium falciparum have been described in the literature. As the screening of a plethora of compounds for antiplasmodial activity is urgently needed and becomes a constantly increasing routine analysis, a test system has to fulfill the following requirements: sensitivity, reliability, simplicity of performance, high-throughput compatibility, and cost-effectiveness. Here, we describe an assay that fulfills all criteria and in which the fluorescent SYTOX Green dye is introduced to determine growth inhibition of Plasmodia in in vitro cultures.     

Separation of modified 2'-deoxyoligonucleotides using ion-pairing reversed-phase HPLC

A group of 18-mers of the same base sequence, but with differing alkyl modifications is used to investigate effects of these modifications on retention of oligonucleotides using ion-pairing reversed-phase liquid chromatography (IP-RPLC). It is shown that IP-RPLC is able to distinguish between oligonucleotides differing only by a single alkyl group. The identity of the nucleobase and position and length of the alkyl adduct affect retention of the oligonucleotide. These separation phenomena result from changes in charge and hydrophobicity upon alkylation. As demonstrated in this paper; chromatographic selectivity, unique to IP-RPLC, greatly facilitates the purification process of modified oligonucleotides.     

Fractionation and identification of proteins by 2-DE and MS: towards a proteomic analysis of Plasmodium falciparum

Since completion of genome sequencing of the malarial parasite Plasmodium falciparum, proteomic tools for the identification of parasite proteins have become particularly attractive as they allow a more thorough interpretation of these data. Recent advances in 2-D PAGE, MS, and bioinformatics have created great opportunities for mapping and characterization of protein populations. We employed these improvements in a proteomic approach for the analysis of proteins detected in two blood stages of P. falciparum, (i) in the schizont stage and (ii) in the merozoite stage. For the isolation of merozoites, we introduced a new protocol based on the preparation of clustered structures of merozoites upon treatment of cultures with the common cysteine proteinase inhibitor E64. Peptide mass fingerprints of excised and trypsinated protein spots, acquired by MALDI-TOF MS were generated to identify a variety of proteins. Moreover, prefractionation procedures were used to enrich and map low-abundance proteins in protein samples. The data demonstrate that classic proteomic analyses using 2-D PAGE are now feasible for P. falciparum and represent the first step in the direction of creating 2-D reference maps for this medically most relevant protozoon.