Novel Biological Substrates of Human Kallikrein 7 Identified Through Degradomics [Genomics and Proteomics]

June 1st, 2015 by Yu, Y., Prassas, I., Dimitromanolakis, A., Diamandis, E. P.

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. While our understanding of the pathophysiological roles of most KLKs has blossomed in recent years, identification of the direct endogenous substrates of human kallikrein-related peptidases (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 (SRM 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 lines, 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.