Beyond Degradation: How Reverse Proteolysis Creates Disease-Relevant Antigens

Proteases are classically viewed as degradative enzymes, yet they can also catalyze reverse proteolysis, a reaction known for decades but rarely examined in disease contexts. Here, we show that cysteine cathepsins efficiently form multi-generational fusion peptides, including chimeras between viral/bacterial and human proteins, revealing a hidden layer of peptidome complexity with direct relevance to autoimmunity. Importantly, we demonstrated that this activity is pH dependent, favored by citrullination, and may represent up to 4.5% of the protease activity. Moreover, we identified cathepsin S-generated type 1 diabetes superagonists, directly linking reverse proteolysis to a clinically established autoantigen. To capture these rare products in a cellular environment, we developed a click chemistry-based technology. These findings expand the protease functional paradigm and uncover a hidden source of disease-relevant antigens.