Different sec-requirements for signal peptide cleavage and protein translocation in a model E. coli protein

AbstractWe describe a secretory E. coli protein with a novel phenotype: signal peptide cleavage is largely unaffected whereas chain translocation is efficiently blocked under conditions where SecA, a central component of the secretory machinery, is rendered non-functional, and we have traced this phenotype to the presence of a mildly hydrophobic segment located ~30 residues downstream of the signal peptide. When this segment is deleted, normal SecA-dependent signal peptide cleavage and chain translocation is observed; when its hydrophobicity is increased, it becomes a permanent membrane anchor with cleavage of the signal peptide and membrane insertion both being SecA-independent. These findings suggest that the initial insertion of the signal peptide across the membrane can be uncoupled from the translocation process proper

A democratically elected president or another Brussels backroom deal? : en utvärdering av förfarandet att utse Europeiska kommissionens ordförande post-Lissabon

One of the latest, and maybe the most innovative, institutional changes in the EU is that the president of the European Commission should be nominated after taking the result of the EP-elections into account. This is said to create a democratic link between the EU-voters and the president, stir EU-wide debate as presidential candidates emerge and hopefully result in higher voter turnout and a president that is “the result of a democratic election”. In this thesis this new procedure is evaluated based on democratic theory. Firstly a democratic model, based on what a procedure of this kind should live up to, is established. The model is then applied to the procedure of 2014 to see if it fulfils the democratic criteria of: publicity, clear definition of mandate, enlightened understanding and political participation. The result was that three of these criteria were not fulfilled. The procedure remained largely unknown among voters, the voters also had limited knowledge of the EU political system necessary to understand the possible outcomes of this procedure and the procedure allowed for almost no individual political participation. Only the third criterion was partly fulfilled. The conclusion is that the procedure therefor will not result in a democratically elected president, nor will it address the problems of democratic deficit or result in a higher voter turnout

Supplementary data for the article: Apostolovic, D.; Sánchez-Vidaurre, S.; Waden, K.; Curin, M.; Grundström, J.; Gafvelin, G.; Cirkovic Velickovic, T.; Grönlund, H.; Thomas, W. R.; Valenta, R.; et al. The Cat Lipocalin Fel d 7 and Its Cross-Reactivity with the Dog Lipocalin Can f 1. Allergy: European Journal of Allergy and Clinical Immunology 2016, 71 (10), 1490–1495. https://doi.org/10.1111/all.12955

Supplementary material for: [https://doi.org/10.1111/all.12955]Related to published version: [http://cherry.chem.bg.ac.rs/handle/123456789/2337]Related to accepted version: [http://cherry.chem.bg.ac.rs/handle/123456789/3650

Cross-reactive EBNA1 immunity targets alpha-crystallin B and is associated with multiple sclerosis

Multiple sclerosis (MS) is an inflammatory disease of the central nervous system, for which and Epstein-Barr virus (EBV) infection is a likely prerequisite. Due to the homology between Epstein-Barr nuclear antigen 1 (EBNA1) and alpha-crystallin B (CRYAB), we examined antibody reactivity to EBNA1 and CRYAB peptide libraries in 713 persons with MS (pwMS) and 722 matched controls (Con). Antibody response to CRYAB amino acids 7 to 16 was associated with MS (OR = 2.0), and combination of high EBNA1 responses with CRYAB positivity markedly increased disease risk (OR = 9.0). Blocking experiments revealed antibody cross-reactivity between the homologous EBNA1 and CRYAB epitopes. Evidence for T cell cross-reactivity was obtained in mice between EBNA1 and CRYAB, and increased CRYAB and EBNA1 CD4$^{+}$ T cell responses were detected in natalizumab-treated pwMS. This study provides evidence for antibody cross-reactivity between EBNA1 and CRYAB and points to a similar cross-reactivity in T cells, further demonstrating the role of EBV adaptive immune responses in MS development

Distant downstream sequence determinants can control N-tail translocation during protein insertion into the Endoplasmic Reticulum membrane

We have studied the membrane insertion of ProW, an Escherichia coli inner membrane protein with seven transmembrane segments and a large periplasmic N-terminal tail, into endoplasmic reticulum (ER)-derived dog pancreas microsomes. Strikingly, significant levels of N-tail translocation is seen only when a minimum of four of the transmembrane segments are present; for constructs with fewer transmembrane segments, the N-tail remains mostly nontranslocated and the majority of the molecules adopt an 'inverted' topology where normally nontranslocated parts are translocated and vice versa. N-tail translocation can also be promoted by shortening of the N-tail and by the addition of positively charged residues immediately downstream of the first trasnmembrane segment. We conclude that as many as four consecutive transmembrane segments may be collectively involved in determining membrane protein topology in the ER and that the effects of downstream sequence determinants may vary depending on the size and charge of the N-tail. We also provide evidence to suggest that the ProW N-tail is translocated across the ER membrane in a C-to-N-terminal direction

Study of polytopic membrane protein topological organization as a function of membrane lipid composition.

A protocol is described using lipid mutants and thiol-specific chemical reagents to study lipid-dependent and host-specific membrane protein topogenesis by the substituted-cysteine accessibility method as applied to transmembrane domains (SCAM). SCAM is adapted to follow changes in membrane protein topology as a function of changes in membrane lipid composition. The strategy described can be adapted to any membrane system

In Vitro Evolution of Allergy Vaccine Candidates, with Maintained Structure, but Reduced B Cell and T Cell Activation Capacity

Allergy and asthma to cat (Felis domesticus) affects about 10% of the population in affluent countries. Immediate allergic symptoms are primarily mediated via IgE antibodies binding to B cell epitopes, whereas late phase inflammatory reactions are mediated via activated T cell recognition of allergen-specific T cell epitopes. Allergen-specific immunotherapy relieves symptoms and is the only treatment inducing a long-lasting protection by induction of protective immune responses. The aim of this study was to produce an allergy vaccine designed with the combined features of attenuated T cell activation, reduced anaphylactic properties, retained molecular integrity and induction of efficient IgE blocking IgG antibodies for safer and efficacious treatment of patients with allergy and asthma to cat. The template gene coding for rFel d 1 was used to introduce random mutations, which was subsequently expressed in large phage libraries. Despite accumulated mutations by up to 7 rounds of iterative error-prone PCR and biopanning, surface topology and structure was essentially maintained using IgE-antibodies from cat allergic patients for phage enrichment. Four candidates were isolated, displaying similar or lower IgE binding, reduced anaphylactic activity as measured by their capacity to induce basophil degranulation and, importantly, a significantly lower T cell reactivity in lymphoproliferative assays compared to the original rFel d 1. In addition, all mutants showed ability to induce blocking antibodies in immunized mice.The approach presented here provides a straightforward procedure to generate a novel type of allergy vaccines for safer and efficacious treatment of allergic patients

Transmembrane protein topology prediction using support vector machines

Background: Alpha-helical transmembrane (TM) proteins are involved in a wide range of important biological processes such as cell signaling, transport of membrane-impermeable molecules, cell-cell communication, cell recognition and cell adhesion. Many are also prime drug targets, and it has been estimated that more than half of all drugs currently on the market target membrane proteins. However, due to the experimental difficulties involved in obtaining high quality crystals, this class of protein is severely under-represented in structural databases. In the absence of structural data, sequence-based prediction methods allow TM protein topology to be investigated.Results: We present a support vector machine-based (SVM) TM protein topology predictor that integrates both signal peptide and re-entrant helix prediction, benchmarked with full cross-validation on a novel data set of 131 sequences with known crystal structures. The method achieves topology prediction accuracy of 89%, while signal peptides and re-entrant helices are predicted with 93% and 44% accuracy respectively. An additional SVM trained to discriminate between globular and TM proteins detected zero false positives, with a low false negative rate of 0.4%. We present the results of applying these tools to a number of complete genomes. Source code, data sets and a web server are freely available from http://bioinf.cs.ucl.ac.uk/psipred/.Conclusion: The high accuracy of TM topology prediction which includes detection of both signal peptides and re-entrant helices, combined with the ability to effectively discriminate between TM and globular proteins, make this method ideally suited to whole genome annotation of alpha-helical transmembrane proteins