In vivo assessment of protease dynamics in cutaneous wound healing by degradomics analysis of porcine wound exudates

Fabio Sabino, Olivia Hermes, Fabian E. Egli, Tobias Kockmann, Pascal Schlage, Pierre Croizat, Jayachandran N. Kizhakkedathu, Hans Smola, Ulrich Auf Dem Keller*

*Corresponding author for this work

Research output: Contribution to journalJournal articleResearchpeer-review

Abstract

Proteases control complex tissue responses by modulating inflammation, cell proliferation and migration, and matrix remodeling. All these processes are orchestrated in cutaneous wound healing to restore the skin's barrier function upon injury. Altered protease activity has been implicated in the pathogenesis of healing impairments, and proteases are important targets in diagnosis and therapy of this pathology. Global assessment of proteolysis at critical turning points after injury will define crucial events in acute healing that might be disturbed in healing disorders. As optimal biospecimens, wound exudates contain an ideal proteome to detect extracellular proteolytic events, are noninvasively accessible, and can be collected at multiple time points along the healing process from the same wound in the clinics. In this study, we applied multiplexed Terminal Amine Isotopic Labeling of Substrates (TAILS) to globally assess proteolysis in early phases of cutaneous wound healing. By quantitative analysis of proteins and protein N termini in wound fluids from a clinically relevant pig wound model, we identified more than 650 proteins and discerned major healing phases through distinctive abundance clustering of markers of inflammation, granulation tissue formation, and re-epithelialization. TAILS revealed a high degree of proteolysis at all time points after injury by detecting almost 1300 N-terminal peptides in ∼450 proteins. Quantitative positional proteomics mapped pivotal interdependent processing events in the blood coagulation and complement cascades, temporally discerned clotting and fibrinolysis during the healing process, and detected processing of complement C3 at distinct time points after wounding and by different proteases. Exploiting data on primary cleavage specificities, we related candidate proteases to cleavage events and revealed processing of the integrin adapter protein kindlin-3 by caspase-3, generating new hypotheses for protease-substrate relations in the healing skin wound in vivo. The data have been deposited to the Proteome- Xchange Consortium with identifier PXD001198.

Original languageEnglish
JournalMolecular and Cellular Proteomics
Volume14
Issue number2
Pages (from-to)354-370
ISSN1535-9476
DOIs
Publication statusPublished - 2015
Externally publishedYes

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