title: Novel Biological Substrates of Human Kallikrein 7 Identified through Degradomics.
abstract: Kallikrein-related peptidases (KLKs) are a group of serine proteases widely expressed in various tissues and involved in a wide range of physiological and pathological processes. Although our understanding of the pathophysiological roles of most KLKs has blossomed in recent years, identification of the direct endogenous substrates of human KLKs remains an unmet objective. In this study we employed a degradomics approach to systemically investigate the endogenous substrates of KLK7 in an effort to understand the molecular pathways underlying KLK7 action in skin. We identified several previously known as well as novel protein substrates. Our most promising candidates were further validated with the use of targeted quantitative proteomics (selected reaction monitoring methods) and in vitro recombinant protein digestion assays. Our study revealed midkine, CYR61, and tenascin-C as endogenous substrates for KLK7. Interestingly, some of these substrates (e.g. midkine) were prone to proteolytic cleavage only by KLK7 (and not by other skin-associated KLKs), whereas others (e.g. CYR61 and tenascin-C) could be digested by several KLKs. Furthermore, using melanoma cell line, we show that KLK7-mediated cleavage of midkine results in an overall reduction in the pro-proliferative and pro-migratory effect of midkine. An inverse relation between KLK7 and midkine is also observed in human melanoma tissues. In summary, our degradomics approach revealed three novel endogenous substrates for KLK7, which may shed more light on the pathobiological roles of KLK7 in human skin. Similar substrate screening approaches could be applied for the discovery of biological substrates of other protease.
authors.affiliation: Department of Laboratory Medicine and Pathobiology, University of Toronto, Toronto, Ontario M5S 1A8, Canada; Department of Pathology and Laboratory Medicine, Mount Sinai Hospital, Toronto, Ontario M5T 3L9, Canada.
authors.forename: Yijing
authors.initials: Y
authors.lastname: Yu
authors.affiliation: Department of Pathology and Laboratory Medicine, Mount Sinai Hospital, Toronto, Ontario M5T 3L9, Canada.
authors.forename: Ioannis
authors.initials: I
authors.lastname: Prassas
authors.affiliation: Department of Pathology and Laboratory Medicine, Mount Sinai Hospital, Toronto, Ontario M5T 3L9, Canada.
authors.forename: Apostolos
authors.initials: A
authors.lastname: Dimitromanolakis
authors.affiliation: Department of Laboratory Medicine and Pathobiology, University of Toronto, Toronto, Ontario M5S 1A8, Canada; Department of Pathology and Laboratory Medicine, Mount Sinai Hospital, Toronto, Ontario M5T 3L9, Canada; Department of Clinical Biochemistry, University Health Network, Toronto, Ontario M5G 2C4, Canada. Electronic address: ediamandis@mtsinai.on.ca.
authors.forename: Eleftherios P
authors.initials: EP
authors.lastname: Diamandis
mesh_terms: D000595:Amino Acid Sequence; D045744:Cell Line, Tumor; D055515:Cysteine-Rich Protein 61 / Q000378:metabolism; D016207:Cytokines / Q000378:metabolism; D006801:Humans; D007610:Kallikreins / Q000737:chemistry / Q000378:metabolism*; D000078792:Midkine; D008969:Molecular Sequence Data; D059748:Proteolysis; D040901:Proteomics; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D013379:Substrate Specificity; D053719:Tandem Mass Spectrometry; D019063:Tenascin / Q000378:metabolism
chemical_list: D055515:Cysteine-Rich Protein 61; D016207:Cytokines; D011994:Recombinant Proteins; D019063:Tenascin; D000078792:Midkine; C082472:KLK7 protein, human; D007610:Kallikreins
keywords: degradomics; kallikrein; protease; proteolysis; proteomics; substrate specificity