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Abstract

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.This review addresses current challenges in revealing the role of proteases in biological processes and presents systematic methodologies for identifying and assigning bona fide substrates to proteases in plants.

Keywords

Activity-based protein profiling probe (ABPP); N-terminomics; plant proteases; protease inhibitors; protease substrates; proteolysis; substrate cleavage site

Published in

Journal of Experimental Botany
2025
Publisher: OXFORD UNIV PRESS

SLU Authors

UKÄ Subject classification

Biochemistry
Botany

Publication identifier

  • DOI: https://doi.org/10.1093/jxb/eraf194

Permanent link to this page (URI)

https://res.slu.se/id/publ/143050