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Aspartic proteases are abundant and active in acidified wound fluid

Barbosa da Silva, E.; Crane, M.; Liu, L.; Gelsinger, D. J.; Jordon, A.; McKinney, R. L.; Eberson, C. P.; Jamieson, A.; O'Donoghue, A. J.

2024-06-30 biochemistry
10.1101/2024.06.30.601389 bioRxiv
Show abstract

Wound healing necessitates a balance between synthesis and breakdown of extracellular matrix components, which is tightly regulated by proteases and their inhibitors. Studies have shown that treatment of poorly healing wounds with acid results in improved healing. In this study, we systematically evaluated changes in proteolytic activity of murine wound fluid upon acidification. A library of 228 synthetic peptides served as reporters of protease activity at pH 7.4, pH 5.0 and pH 3.5. The peptide digestion patterns differed at each pH, revealing that proteases active at pH 7.4 are inactivated at pH 3.5. Notably, aspartic acid proteases emerged as the dominant active enzymes at pH 3.5 and their activity was inhibited by pepstatin. Using a fluorogenic substrate, we quantified aspartic protease activity across varying pH levels and demonstrated optimal activity between pH 3.0 and 3.8. This activity was detectable as early as one day post-injury and persisted over the following ten days. Importantly, human wound fluid exhibited the same activity profile, validating the mouse model as a relevant system for studying acid-mediated wound healing processes.

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