Four guidelines to identify bona fide protease-substrate pairs

Plant proteases are an important class of enzymes, with proposed involvement in various aspects of the plant life cycle. However, pinpointing authentic protease-substrate interactions remains challenging, which hinders a comprehensive understanding of the biological function of proteases. Moreover, a structured set of guidelines to validate protease substrates is lacking. In this review, we outline a minimum of four key guidelines that, when followed, can confirm the specificity of protease-substrate interaction for proteases that perform limited proteolysis and with specific cleavage sites: (i) the observation of substrate cleavage; (ii) the reduction in substrate cleavage due to protease inhibitors or (iii) genetic mutation of the protease; and (iv) a final proof of the specificity of the substrate cleavage site. It is important to emphasize that these guidelines are not universally applicable to all proteases. By creating a set of guidelines, summarizing current findings and proposing future research directions, this review aims to highlight innovative techniques that will improve the specificity and accuracy of protease research and facilitate a deeper understanding of the role of proteases in plant biology.