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Matrix metalloproteinase degradomics at the epidermal-dermal interface

Final Report Summary - SKINTERMINOMICS (Matrix metalloproteinase degradomics at the epidermal-dermal interface)

Matrix metalloproteinases (MMPs) are secreted enzymes that can degrade proteins of the cellular microenvironment, the extracellular matrix (ECM), and thereby allow migration of skin cells to facilitate wound closure but also tumor invasion. Recent research showed that MMPs also specifically cleave bioactive proteins, such as chemokines, and thereby regulate the recruitment of immune cells to sites of inflammation in wound healing and carcinogenesis. This is of crucial importance for wound repair and also for the pathogenesis of skin cancer. One member of the MMP family - MMP10 - gained particular interest, since it is specifically secreted by hyperproliferative keratinocytes and in close proximity to stromal fibroblasts in wounds and in skin tumors. Therefore, modifying MMP10 activity might have therapeutic effects for the treatment of chronic wounds and cancer. However, there is not much known about MMP10 function, besides its specific expression in proliferating epithelia and potential implication in cell migration and tissue invasion. The key to unravel the mechanism of MMP10 action and to evaluate this protease as a therapeutic target is the system-wide identification of its target proteins (substrates) under physiological and pathological conditions. In recent years, several powerful mass spectrometry-based technologies have been developed to identify these protease substrates in complex biological mixtures and under physiological conditions. One of these techniques, Terminal Amine Isotopic Labeling of Substrates (TAILS), is particularly suited for the concomitant analysis of multiple conditions within the same experiment.

In this project, we exploited this specific advantage and modified TAILS to not only identify new substrates but also to monitor their cleavage over time. This significantly enhanced confidence in protease substrate discovery and helped to categorize cleavages based on clusters of processing events with different efficiencies. Next, we applied this improved technology to identify new MMP10 substrates in cell culture supernatants from keratinocytes and fibroblasts. Thereby, we identified novel MMP10-dependent cleavages of extracellular matrix proteins like type I collagen and ADAMTS-like protein 1 (ADAMTSL1) as well as of growth factor receptors, such as platelet-derived growth factor receptor alpha (PDGFRalpha). The functional importance of these cleavages for cellular behavior in healing skin wounds and skin tumors are currently under further investigation.

The second objective of this project aimed to better understand proteolysis under conditions of epidermal hyperproliferation. For this purpose, we used a mouse model of cutaneous wound healing and employed TAILS to examine protease cleavage products during skin repair at important steps of the healing process. Thereby, we related highly abundant proteases to cleavages at distinct time points after wounding and are currently investigating the functions of specific protease-substrate relations.

In conclusion, this project used and further developed the newest technologies to generate novel hypotheses for the elucidation of protease function in hyperproliferative epithelia. This opened up several avenues for future research, which are currently explored and will help to further explore proteases as drug targets to address unmet medical needs.