Abstract
Numerous studies highlight the fact that concerted proteolysis is essential for skin morphology and function. The cysteine protease cathepsin L (Ctsl) has been implicated in epidermal proliferation and desquamation, as well as in hair cycle regulation. In stark contrast, mice deficient in cathepsin B (Ctsb) do not display an overt skin phenotype. To understand the systematic consequences of deleting Ctsb or Ctsl, we determined the protein abundances of >1300 proteins and proteolytic cleavage events in skin samples of wild-type, Ctsb(-/-), and Ctsl(-/-) mice via mass-spectrometry-based proteomics. Both protease deficiencies revealed distinct quantitative changes in proteome composition. Ctsl(-/-) skin revealed increased levels of the cysteine protease inhibitors cystatin B and cystatin M/E, increased cathepsin D, and an accumulation of the extracellular glycoprotein periostin. Immunohistochemistry located periostin predominantly in the hypodermal connective tissue of Ctsl(-/-) skin. The proteomic identification of proteolytic cleavage sites within skin proteins revealed numerous processing sites that are underrepresented in Ctsl(-/-) or Ctsb(-/-) samples. Notably, few of the affected cleavage sites shared the canonical Ctsl or Ctsb specificity, providing further evidence of a complex proteolytic network in the skin. Novel processing sites in proteins such as dermokine and Notch-1 were detected. Simultaneous analysis of acetylated protein N termini showed prototypical mammalian N-alpha acetylation. These results illustrate an influence of both Ctsb and Ctsl on the murine skin proteome and degradome, with the phenotypic consequences of the absence of either protease differing considerably.
Highlights
From the ‡Institute of Molecular Medicine and Cell Research, University of Freiburg, D-79104 Freiburg, Germany; §Faculty of Biology, University of Freiburg, D-79104 Freiburg, Germany; ¶Spemann Graduate School of Biology and Medicine (SGBM), University of Freiburg, D-79104 Freiburg, Germany; ʈLaboratory of Tumor and Development Biology, CHU, GIGA-Cancer, University of Liege, B-4000 Liege, Belgium; **Department of Pathology and Laboratory Medicine and Department of Chemistry, Centre for Blood Research, University of British Columbia, Vancouver, British Columbia, Canada V6T 1Z3; ‡‡Freiburg Institute for Advanced Studies (FRIAS), School of Life Science-LifeNet, University of Freiburg, D-79104 Freiburg, Germany; §§ZBSA Center for Biological Systems Analysis, University of Freiburg, D-79104 Freiburg, Germany; ¶¶BIOSS Centre for Biological Signaling Studies, University of Freiburg, D-79104 Freiburg, Germany
S1A and S1B) and an average fold change (Fc) close to 0 (Fig. 1B). Both cathepsin B (Ctsb) and cathepsin L (Ctsl) had a pronounced impact on the skin proteome composition. 1191 proteins were identified in both Ctsb replicates, and 1317 proteins were identified in both Ctsl replicates. 356 proteins were changed more than 2-fold (Fc Ͻ Ϫ0.58 or Fc Ͼ 0.58) in both Ctsb replicates, and 517 proteins were changed more than 2-fold in both Ctsl replicates
Increased levels of active cathepsin D (Ctsd) have been found in CtslϪ/Ϫ MEFs [59] and, most notably, in the brains of Ctsb/Ctsl double deficient mice [25, 26]
Summary
Cathepsin B; Ctsd, cathepsin D; Ctsl, cathepsin L; E64, trans-epoxysuccinyl-L-leucylamido(4-guanidino)butane; Fc, fold change; GFP, green fluorescent protein; LC-MS/ MS, liquid chromatography–tandem mass spectrometry; MEF, mouse embryonic fibroblast; TAILS, terminal amine isotopic labeling of substrates. Cathepsin B (Ctsb)-gene-deficient mice do not display a spontaneous phenotype [18, 19], but if pathologically challenged these mice are less susceptible to disease in pancreatitis [20] and are less affected by TNF␣-induced hepatocyte apoptosis [21]. We performed terminal amine isotopic labeling of substrates (TAILS) [29] to determine changes in the skin proteome cleavage pattern and to identify Ctsb- and Ctsl-dependent cleavage events. Our findings highlight the pivotal function of these proteases in maintaining proteome homeostasis and in balancing the proteolytic network This is one of the first studies investigating how the deletion of individual proteases affects proteolytic processing in vivo
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