Immunoproteasome LMP2 60HH Variant Alters MBP Epitope Generation and Reduces the Risk to Develop Multiple Sclerosis in Italian Female Population

Background

Albeit several studies pointed out the pivotal role that CD4+T cells have in Multiple Sclerosis, the CD8+ T cells involvement in the pathology is still in its early phases of investigation. Proteasome degradation is the key step in the production of MHC class I-restricted epitopes and therefore its activity could be an important element in the activation and regulation of autoreactive CD8+ T cells in Multiple Sclerosis.

Methodology/Principal Findings

Immunoproteasomes and PA28-αβ regulator are present in MS affected brain area and accumulated in plaques. They are expressed in cell types supposed to be involved in MS development such as neurons, endothelial cells, oligodendrocytes, macrophages/macroglia and lymphocytes. Furthermore, in a genetic study on 1262 Italian MS cases and 845 controls we observed that HLA-A*02+ female subjects carrying the immunoproteasome LMP2 codon 60HH variant have a reduced risk to develop MS. Accordingly, immunoproteasomes carrying the LMP2 60H allele produce in vitro a lower amount of the HLA-A*0201 restricted immunodominant epitope MBP_111–119.

Conclusion/Significance

The immunoproteasome LMP2 60HH variant reduces the risk to develop MS amongst Italian HLA-A*02+ females. We propose that such an effect is mediated by the altered proteasome-dependent production of a specific MBP epitope presented on the MHC class I. Our observations thereby support the hypothesis of an involvement of immunoproteasome in the MS pathogenesis.     

Engineering receptors and antibodies for biosensors

Biosensor sensitivity and selectivity depend essentially on the properties of the biorecognition elements to be used for analyte binding. Two principally different applications are considered, (1) effects monitoring with biological components as targets for bioeffective substances, among them endocrine disruptors; and (2) immunochemical analysis employing antibodies as binding proteins for a wide variety of analytes such as pesticides. Genetic engineering provides an elegant way not only for providing unlimited amounts of biorecognition molecules but also for the alteration of existing properties and the supplementation with additional functions. Instrumental applications were carried out with the optical sensor BIAcore. The first example deals with the characterization of receptors. For this purpose, the human estrogen receptor alpha was used. Binding studies were carried out with natural as well as xenoestrogens. An equilibrium dissociation constant K(d) of 2.3x10(-10) (M) was derived for 17beta-estradiol. A competition assay was performed with a bovine serum albumin (BSA)-17beta-estradiol conjugate, immobilized at the optical sensor surface, and the free estrogen. The signals obtained represent estradiol equivalents. This format was transferred to a microplate-based enzyme-linked receptor assay. It reached a detection limit of 0.02 microg l(-1) 17beta-estradiol and proved suitable for the detection of natural and synthetic estrogens as well as xenoestrogens in field studies. The second example is targeted at kinetic and affinity measurements of recombinant antibody fragments derived from antibody libraries with s-triazine selectivities. Different strategies for the synthesis of antibody fragment libraries, followed by the selection of specific antibody variants, were examined. An antibody library was derived from a set of B cells. Chain shuffling of the heavy and light chains provided the best binders. An enzyme linked immunosorbent assay (ELISA) was achieved for atrazine with an IC(50) of 0.9 microg l(-1) and a detection limit of 0.2 microg l(-1). The close relations between the optimization of recombinant antibodies by evolutionary strategies and genetic algorithms are considered.     

Evaluation of a sensitive detection method for peptide arrays prepared by SPOT synthesis

The growing range of applications for peptide arrays prepared by SPOT synthesis confirms that they are a powerful proteomics technique to study numerous aspects of molecular interaction events. The most frequent application for peptide arrays prepared by SPOT synthesis is the identification of linear epitopes that are recognized by antibodies. In the conventional format using secondary antibodies for detection unspecific binding and high background have been observed. This leads to difficulties in evaluation of developed membranes. Especially for application with combinatorial libraries false positive results are to be avoided. To circumvent this issue, we directly labeled compounds of interest with biotin and detected binding by incubation with streptavidin-horseradish peroxidase via chemiluminescence. Optimization of method conditions led to a very sensitive detection technique with no or low number of unspecific spots, which is superior to conventional detection with secondary antibodies. As one consequence, evaluation of competitive assays got more reliable.     

A quantitative analysis of spontaneous isoaspartate formation from N-terminal asparaginyl and aspartyl residues

The formation of isoaspartate (isoAsp) from asparaginyl or aspartyl residues is a spontaneous post-translational modification of peptides and proteins. Due to isopeptide bond formation, the structure and possibly function of peptides and proteins is altered. IsoAsp modifications within the peptide chain have been reported for many cytosolic proteins. Amyloid peptides (Aβ) deposited in Alzheimer’s disease may carry an N-terminal isoAsp-modification. Here, we describe a quantitative investigation of isoAsp-formation from N-terminal Asn and Asp using model peptides similar to the Aβ N-terminus. The study is based on a newly developed separation of peptides using capillary electrophoresis (CE). ¹H NMR was employed to validate the basic finding of N-terminal isoAsp-formation from Asp and Asn. Thereby, the isomerization of Asn at neutral pH (0.6 day^?1, peptide NGEF) is approximately six times faster than that within the peptide chain (AANGEF). The difference in velocity between Asn and Asp isomerization is approximately 50-fold. In contrast to N-terminal Asn, Asp isomerization is significantly accelerated at acidic pH. The kinetic solvent isotope (k _D2O/k _H2O) effect of 2.46 suggests a rate-limiting proton transfer in isoAsp-formation. The proton inventory is consistent with transfer of one proton in the transition state, supporting the previous notion of rate-limiting deprotonation of the peptide backbone amide during succinimide-intermediate formation. The study provides evidence for a spontaneous N-terminal isoAsp-formation within peptides and might explain the accumulation of N-terminal isoAsp in amyloid deposits.     

Subsurface microbial methanotrophic mats in the Black Sea

A nodule-shaped microbial mat was found subsurface in sediments of a gas seep in the anoxic Black Sea. This mat was dominated by ANME-1 archaea and consumed methane and sulfate simultaneously. We propose that such subsurface mats represent the initial stage of previously investigated microbial reefs.     

The antibacterial activity of 4,4'-bipyridinium amphiphiles with conventional, bicephalic and gemini architectures

Dialkyl 4,4'-bipyridinium compounds are widely employed for their useful redox properties, and are commonly known as viologens due to their intense coloration upon reduction. Despite their prevalence and amphiphilic nature, the antibacterial activity of these compounds remains largely unreported. We have thus prepared a series of mono- and bis-alkylated analogs of 4,4'-bipyridine to investigate structure-activity relationships in their inhibition of a battery of Gram positive and Gram negative bacteria. The prepared cationic compounds were conventional (one cationic head, one non-polar tail), bicephalic (two heads, one tail), or gemini (two heads, two tails) in their amphiphilic structure. Additionally, an isomeric series of six bis-alkylated compounds ranging from symmetric (PQ-11,11) to highly asymmetric (PQ-20,2) were prepared. Four themes of bioactivity emerged: (1) the most bioactive compounds were gemini in structure; (2) 22 carbons in the alkyl chains, with little to modest asymmetry, led to optimal activity; (3) bicephalic compounds were generally comparable to conventional amphiphiles, though only about 12 carbons in the alkyl chains were solubilized in water by each cationic nitrogen; (4) the effects of counterion identity were not evident between chlorides and bromides; however, the presence of the iodide counterion inhibited dissolution in all compounds tested. Three isomeric compounds with little to no asymmetry in tail length, PQ-11,11, PQ-12,10, and PQ-14,8, prepared as the bromide salts, showed comparable and highly potent activity, with MIC levels around 2 μM against 3 of 4 bacteria tested. The simple (one- to two-step) syntheses of potent antimicrobials portend well for future optimization.