Abstract
Matrix metalloproteinases (MMPs) are important players in skin homeostasis, wound repair, and in the pathogenesis of skin cancer. It is now well established that most of their functions are related to processing of bioactive proteins rather than components of the extracellular matrix (ECM). MMP10 is highly expressed in keratinocytes at the wound edge and at the invasive front of tumors, but hardly any non-ECM substrates have been identified and its function in tissue repair and carcinogenesis is unclear. To better understand the role of MMP10 in the epidermis, we employed multiplexed iTRAQ-based Terminal Amine Isotopic Labeling of Substrates (TAILS) and monitored MMP10-dependent proteolysis over time in secretomes from keratinocytes. Time-resolved abundance clustering of neo-N termini classified MMP10-dependent cleavage events by efficiency and refined the MMP10 cleavage site specificity by revealing a so far unknown preference for glutamate in the P1 position. Moreover, we identified and validated the integrin alpha 6 subunit, cysteine-rich angiogenic inducer 61 and dermokine as novel direct MMP10 substrates and provide evidence for MMP10-dependent but indirect processing of phosphatidylethanolamine-binding protein 1. Finally, we sampled the epidermal proteome and degradome in unprecedented depth and confirmed MMP10-dependent processing of dermokine in vivo by TAILS analysis of epidermis from transgenic mice that overexpress a constitutively active mutant of MMP10 in basal keratinocytes. The newly identified substrates are involved in cell adhesion, migration, proliferation, and/or differentiation, indicating a contribution of MMP10 to local modulation of these processes during wound healing and cancer development. Data are available via ProteomeXchange with identifier PXD002474.
Highlights
Matrix metalloproteinases (MMPs)1 are extracellular zincdependent endoproteinases that are highly expressed in tissues undergoing remodeling processes during development, in response to injury, or as a result of neoplastic transformation [1,2,3]
Because MMP10 has been mostly neglected in the quest for new MMP substrates, it remains to be elucidated, if it exerts its functions in part by processing of bioactive proteins whose identification is instrumental in understanding the mechanisms of action of MMP10 in tissue repair and carcinogenesis
We employed iTRAQ-Terminal Amine Isotopic Labeling of Substrates (TAILS) on in vitro and in vivo samples to identify substrates of MMP10 that is highly expressed in migrating/invasively growing keratinocytes of skin wounds and cancers (4 – 6)
Summary
Cell Growth and Preparation of Secreted Proteins—MMP10 knockout mice were obtained from the Mutant Mouse Regional Resource Center (MMRRC) at UC Davis (strain# 011737-UCD). Secretomes of MMP10-deficient keratinocytes were incubated with active MMP10 at an enzyme/protein ratio of 1:170 (w/w) in the presence of 10 mM CaCl2 and 100 mM NaCl for up to 16 h at 37 °C. For 4plex-iTRAQ-TAILS analysis proteins were precipitated by addition of six volumes of ice-cold acetone (4 h at Ϫ20 °C), pelleted by centrifugation (13,000 rpm, 30 min, 4 °C) and resuspended at a concentration of 1 mg/ml in TAILS buffer (2.5 M GuHCl, 250 mM HEPES, pH 7.8). Protein and peptide quantification in epidermal lysates (4plex) was performed as described [37] and log2-ratios calculated from iTRAQ reporter ion intensities upon quantile normalization. MS/MS spectra for Nterminal peptides are supplied as Supplemental Data
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