The S phase checkpoint promotes the Smc5/6 complex dependent SUMOylation of Pol2, the catalytic subunit of DNA polymerase ε

Replication fork stalling and accumulation of single-stranded DNA trigger the S phase checkpoint, a signalling cascade that, in budding yeast, leads to the activation of the Rad53 kinase. Rad53 is essential in maintaining cell viability, but its targets of regulation are still partially unknown. Here we show that Rad53 drives the hyper-SUMOylation of Pol2, the catalytic subunit of DNA polymerase ε, principally following replication forks stalling induced by nucleotide depletion. Pol2 is the main target of SUMOylation within the replisome and its modification requires the SUMO-ligase Mms21, a subunit of the Smc5/6 complex. Moreover, the Smc5/6 complex co-purifies with Pol ε, independently of other replisome components. Finally, we map Pol2 SUMOylation to a single site within the N-terminal catalytic domain and identify a SUMO-interacting motif at the C-terminus of Pol2. These data suggest that the S phase checkpoint regulate Pol ε during replication stress through Pol2 SUMOylation and SUMO-binding abilit

Optimisation and use of humanised RBL NF-AT-GFP and NF-AT-DsRed reporter cell lines suitable for high-throughput scale detection of allergic sensitisation in array format and identification of the ECM–Integrin interaction as critical factor

We have previously described a microarray platform combining live basophils with protein arrays suitable for high-throughput detection of sensitisation against allergens. During optimisation of this technique, we observed severe losses of adhering cells during the washing steps, particularly after activation. In order to preserve cell binding, we tested the cell adhesion characteristics of different extracellular matrix proteins: human collagen I, fibronectin (FN) from bovine plasma and laminin (LN). FN was more effective than LN and collagen. Cell detachment after activation was in part due to reduced surface expression of VLA-4, the main ligand for FN, which was significantly decreased within 15 min of stimulation with 1 μg/mL calcium ionophore A23187, reaching a minimum after 2 h then slowly recovering. These optimised conditions were used for testing of well-characterised sera from allergic patients using two newly developed rat basophil leukaemia stable reporter cell lines (RBL NF-AT/GFP and RBL NF-AT/DsRed), which both express the human high-affinity IgE receptor alpha chain (FcεRIα). Both cell lines were able to detect sensitisation to specific allergens showing the expected bell-shaped dose–response curve, and correlated (R 2 = 0.75) with the standard beta-hexosaminidase assay, which is not suitable for an array format

Further studies on the biological activity of hazelnut allergens

BACKGROUND Sensitization to hazelnut allergens vary depending on the geographic origin and age of the patients. The objective of this study was to further investigate the allergenic activity of hazelnut allergens using sera from patients recruited in various European regions and presenting different sensitization patterns to hazelnut proteins. METHODS Natural Cor a 11 and Cor a 9 were purified from hazelnut whereas Cor a 1 and Cor a 8 were produced as recombinant proteins (rCor a 1.04 and rCor a 8). Sera from hazelnut allergic patients were collected in France (n = 5), Switzerland (n = 2), Greece (n = 11) and Spain (n = 3), within the Europrevall project. Total and allergen-specific IgE were quantified by enzyme allergosorbent test and IgE immunoblot were performed using pooled sera from birch-pollen endemic region or from Greece. Histamine Release (HR) assays were performed with stripped basophils passively sensitized with individual sera and challenged by a hazelnut extract or the different hazelnut allergens. RESULTS As previously described, hazelnut allergic patients from Mediterranean countries are mainly sensitized to the nsLTP Cor a 8 whereas patients from France and Switzerland are sensitized to pollen-related allergens. Interestingly, an intermediate profile was evidenced in patients from Madrid. Hazelnut 7S globulin (Cor a 11) and 11S globulin (Cor a 9) were found to be minor allergens, recognized only by patients from Mediterranean countries. The biologic activity of the 4 tested allergens, analysed by HR assay, further confirmed the sensitization patterns, but also demonstrated the very high elicitation potency of Cor a 8. CONCLUSIONS This work, extending previously published researches, represents a step towards the better understanding of the complexity of hazelnut allergy and provides new data on the biological activity of hazelnut allergens and extracts