Understanding the function of the thousands of cellular proteins is a central question in molecular cell biology. As proteins are typically part of multiple dynamic and often overlapping macromolecular complexes exerting distinct functions, the identification of protein–protein interactions (PPI) and their assignment to specific complexes is a crucial but challenging task. We present a protein fragments complementation assay integrated with the proximity-dependent biotinylation technique BioID. Activated on the interaction of two proteins, split-BioID is a conditional proteomics approach that allows in a single and simple assay to both experimentally validate binary PPI and to unbiasedly identify additional interacting factors. Applying our method to the miRNA-mediated silencing pathway, we can probe the proteomes of two distinct functional complexes containing the Ago2 protein and uncover the protein GIGYF2 as a regulator of miRNA-mediated translation repression. Hence, we provide a novel tool to study dynamic spatiotemporally defined protein complexes in their native cellular environment.

Background: SARS-CoV-2 vaccination in cancer patients is crucial to prevent severe COVID-19 disease course. Methods: This study assessed immunogenicity of cancer patients on active treatment receiving mRNA-based SARS-CoV-2 vaccine by detection of anti-SARS-CoV-2 S1 IgG antibodies in serum, before, after the first and second doses and 3 months after a complete primary course of vaccination. Results were compared with healthy controls. Results: Of 112 patients, the seroconversion rate was 96%. A significant reduction in antibody levels was observed 3 months after vaccination in patients receiving immune checkpoint inhibitors versus control participants (p < 0.001). Adverse events were mostly mild. Conclusion: Immunogenicity after mRNA-based vaccine in cancer patients is adequate but influenced by the type of anticancer therapy. Antibody levels decline after 3 months, and thus a third vaccination is warranted.

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Background: Clinical and experimental analyses indicate a pathognomonic role for allergen IgE crosslinking through epitope-paratope interactions as a major initial step in the cascade leading to effector cell activation and clinical manifestations of lgE-mediated food allergies. We aimed to undertake the initial development and assessment of Ara h 2-specific IgE epitope-like peptides that can bind to allergenspecific IgE paratopes and suppress effector cell activation. Methods:We performed biopanning, screening, IgE binding, selection and mapping of peptides. We generated synthetic peptides for use in all functional experiments. ImmunoCAP inhibition, basophil and mast cell activation tests, with LAD2 cells, a human mast cell line were performed. Twenty-six children or young adults who had peanut allergy were studied.Results: We identified and selected three linear peptides (DHPRFNRDNDVA, DHPRYGP and DHPRFST), and immunoblot analyses revealed binding to lgE from peanut-allergic individuals. The peptide sequences were aligned to the disordered region corresponding to the loop between helices 2 and 3 of Ara h 2, and conformational mapping showed that the peptides match the surface of Ara h 2 and h 6 but not other peanut allergens. In ImmunoCAP inhibition experiments, the peptides significantly inhibit the binding of IgE to Ara h 2 (p < .001). In basophil and mast cell activation tests, the peptides significantly suppressed Ara h 2-induced effector cell activation (p < .05) and increased the half-maximal Ara h 2 effective concentration (p < .05). Binding of the peptides to specific IgEs did not induce activation of basophils or mast cells.Conclusions: These studies show that the indicated peptides reduce the allergenic activity of Ara h 2 and suppress lgE-dependent basophil and mast cell activation.These observations may suggest a novel therapeutic strategy for food allergy based on epitope-paratop blocking.

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