Substrate Profiling of Cysteine Proteases in Physiology and Pathology

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Proteases mold proteomes through limited, site-specific protein processing and protein degradation. For most proteases almost no in vivo substrates are known. For cysteine cathepsins, this contrasts an expanding list of physiological and pathological functions, including epidermal homeostasis (cathepsin L) and tumorigenesis (cathepsin B). Through system-wide substrate profiling, we provide the molecular basis for cathepsin function. Along with active site specificity determination, we identify substrates and cleavage sites in complex proteomes by liquid chromatography/tandem mass spectrometry based strategies. Cathepsin-dependent protein degradation is examined by time-resolved pulse-chase strategies. Experimental systems for substrate profiling include murine models of cathepsin biology in skin, liver, and tumor context with in vitro, cell contextual, and in vivo assays. Substrate candidates undergo multi-stage validation including cleavage kinetics, in situ co-localization of protease and substrates, and functional characterization of processed substrates in the cellular context. We reconstruct proteolytic pathways in vivo by transgenic expression of altered or processed substrates in protease- and substrate-deficient cells. In summary, we establish a platform for protease substrate profiling with the aim to explore new perspectives in protease biology.