title: In vivo assessment of protease dynamics in cutaneous wound healing by degradomics analysis of porcine wound exudates.
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 ProteomeXchange Consortium with identifier PXD001198.
authors.affiliation: From the ‡ETH Zurich, Department of Biology, Institute of Molecular Health Sciences, Otto-Stern-Weg 7, 8093 Zurich, Switzerland;
authors.forename: Fabio
authors.initials: F
authors.lastname: Sabino
authors.affiliation: From the ‡ETH Zurich, Department of Biology, Institute of Molecular Health Sciences, Otto-Stern-Weg 7, 8093 Zurich, Switzerland;
authors.forename: Olivia
authors.initials: O
authors.lastname: Hermes
authors.affiliation: From the ‡ETH Zurich, Department of Biology, Institute of Molecular Health Sciences, Otto-Stern-Weg 7, 8093 Zurich, Switzerland;
authors.forename: Fabian E
authors.initials: FE
authors.lastname: Egli
authors.affiliation: From the ‡ETH Zurich, Department of Biology, Institute of Molecular Health Sciences, Otto-Stern-Weg 7, 8093 Zurich, Switzerland;
authors.forename: Tobias
authors.initials: T
authors.lastname: Kockmann
authors.affiliation: From the ‡ETH Zurich, Department of Biology, Institute of Molecular Health Sciences, Otto-Stern-Weg 7, 8093 Zurich, Switzerland;
authors.forename: Pascal
authors.initials: P
authors.lastname: Schlage
authors.affiliation: §Paul Hartmann AG, Paul Hartmann Strasse 12, 89522 Heidenheim, Germany;
authors.forename: Pierre
authors.initials: P
authors.lastname: Croizat
authors.affiliation: ¶University of British Columbia, Department of Pathology and Laboratory Medicine and Department of Chemistry, Centre for Blood Research, 4.401 Life Sciences Institute, 2350 Health Sciences Mall, Vancouver, British Columbia, Canada V6T 1Z3.
authors.forename: Jayachandran N
authors.initials: JN
authors.lastname: Kizhakkedathu
authors.affiliation: §Paul Hartmann AG, Paul Hartmann Strasse 12, 89522 Heidenheim, Germany;
authors.forename: Hans
authors.initials: H
authors.lastname: Smola
authors.affiliation: From the ‡ETH Zurich, Department of Biology, Institute of Molecular Health Sciences, Otto-Stern-Weg 7, 8093 Zurich, Switzerland; ulrich.aufdemkeller@biol.ethz.ch.
authors.forename: Ulrich
authors.initials: U
authors.lastname: auf dem Keller
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D053148:Caspase 3 / Q000378:metabolism; D002460:Cell Line; D003167:Complement Activation; D003176:Complement C3 / Q000378:metabolism; D005122:Exudates and Transudates / Q000378:metabolism*; D005260:Female; D005342:Fibrinolysis; D006801:Humans; D007553:Isotope Labeling; D008954:Models, Biological; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000737:chemistry / Q000378:metabolism; D010447:Peptide Hydrolases / Q000378:metabolism*; D059748:Proteolysis; D020543:Proteome / Q000378:metabolism; D040901:Proteomics / Q000379:methods*; D012867:Skin / Q000378:metabolism* / Q000473:pathology*; D034421:Sus scrofa; D014945:Wound Healing
chemical_list: D003176:Complement C3; D010446:Peptide Fragments; D020543:Proteome; D010447:Peptide Hydrolases; D053148:Caspase 3