Cold shock Y-box binding protein-1 participates in cancer cell transformation and mediates invasive cell growth. It is unknown whether an autoimmune response against cancerous human YB-1 with posttranslational protein modifications or processing develops. We performed a systematic analysis for autoantibody formation directed against conformational and linear epitopes within the protein. Full-length and truncated recombinant proteins from prokaryotic and eukaryotic cells were generated. Characterization revealed a pattern of spontaneous protein cleavage, predominantly with the prokaryotic protein. Autoantibodies against prokaryotic, but not eukaryotic full-length and cleaved human YB-1 protein fragments were detected in both, healthy volunteers and cancer patients. A mapping of immunogenic epitopes performed with truncated E. coli-derived GST-hYB-1 proteins yielded distinct residues in the protein N- and C-terminus. A peptide array with consecutive overlapping 15mers revealed six distinct antigenic regions in cancer patients, however to a lesser extent in healthy controls. Finally, a protein cleavage assay was set up with recombinant pro- and eukaryotic-derived tagged hYB-1 proteins. A distinct cleavage pattern developed, that is retarded by sera from cancer patients. Taken together, a specific autoimmune response against hYB-1 protein develops in cancer patients with autoantibodies targeting linear epitopes.

DNA-binding protein A (DbpA) belongs to the Y-box family of cold shock domain (CSD) proteins that bind RNA/DNA and exert intracellular functions in cell stress, proliferation, and differentiation. Given the pattern of DbpA staining in inflammatory glomerular diseases, without adherence to cell boundaries, we hypothesized extracellular protein occurrence and specific functions. Lipopolysaccharide and ionomycin induce DbpA expression and secretion from melanoma and mesangial cells. Unlike its homologue Y-box-binding protein 1 (YB-1), DbpA secretion requires inflammasome activation, as secretion is blocked upon the addition of a NOD-like receptor protein-3 (NLRP3) inhibitor. The addition of recombinant DbpA enhances melanoma cell proliferation, migration, and competes with tumor necrosis factor (TNF) binding to its receptor (TNFR1). In TNF-induced cell death assays, rDbpA initially blocks TNF-induced apoptosis, whereas at later time points (>24 h), cells are more prone to die. Given that CSD proteins YB-1 and DbpA fulfill the criteria of alarmins, we propose that their release signals an inherent danger to the host. Some data hint at an extracellular complex formation at a ratio of 10:1 (DbpA:YB-1) of both proteins.