Deletion of Cysteine Cathepsins B or L Yields Differential Impacts on Murine Skin Proteome and Degradome

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 Cathepsins B and L are ubiquitously expressed papain-like cysteine proteases belonging to the C1a papain family (clan CA), with 11 members in humans (1) and 18 members in mice (2). Most cysteine cathepsins like cathepsin L are endopeptidases, whereas cathepsin B shows both endopeptidase and carboxydipeptidase activity (3). Mainly localized in the endosomal/lysosomal compartment, cathepsins have traditionally been thought to play important roles in lysosomal protein turnover. Additional specific functions have been postulated that link cathepsins to different physiological and pathological processes.Studies using cathepsin L (Ctsl)-gene-deficient mice 1 revealed an important role of Ctsl in cardiac homeostasis (4 -6) and a contribution of Ctsl to MHC II-mediated antigen presentation (7, 8) and prohormone processing (9, 10). In a mouse model of pancreatic neuroendocrine cancer, Ctsl promoted tumor growth and invasiveness (11,12). In stark contrast, Ctsl was found to attenuate tumor progression in mouse models of skin cancer, highlighting the context-specific function of this protease (13,14).The most prominent phenotype of Ctsl-deficient mice is periodic hair loss together with epidermal hyperplasia, acanthosis, and hyperkeratosis (15). These alterations in skin morphology are assumed to be keratinocyte specific, as controlled re-expression of Ctsl under a keratin 14 promoter Research

Matrix Metalloproteinase 10 Degradomics in Keratinocytes and Epidermal Tissue Identifies Bioactive Substrates With Pleiotropic Functions*

“…Moreover, because of the significantly higher complexity of the sample, TAILS presumably missed many substrate cleavages in epidermal lysates that had been identified in the cell-based screen. This is in agreement with previous studies, which analyzed cathepsins or MMPs using in vitro and in vivo systems (22,47,77,85). Finally, many substrates might only be accessible to MMP10-dependent processing at the very tip of the wound epidermis or the invasive front of tumors, when keratinocytes are exposed to proliferative and migratory stimuli and an altered extracellular environment (78,86).…”

“…With more than 2000 proteins and almost 3000 N termini we recorded the most comprehensive epidermal proteome and N-terminome reported to date and specifically increased coverage of the epidermis compared with previous studies that analyzed lysates from whole skin (22,77). The only very minor quantitative effects of increased abundance of MMP10 on proteins in the epidermis could be attributed to expression of the transgene in only a small fraction of the cells contributing to the epidermal lysates, but they also reflect the robustness of the proteolytic network in the skin that was also not dramatically affected by loss of individual cathepsins or MMP2 under unchallenged conditions (22,77). Moreover, because of the significantly higher complexity of the sample, TAILS presumably missed many substrate cleavages in epidermal lysates that had been identified in the cell-based screen.…”

“…We propose that proteolytic processing provides an additional level of regulation for this potential soluble mediator. Indeed, processing of DMKN by cathepsin B in the skin has recently been reported (77). Notably, cleavage of DMKN by MMP10 did not obviously impact epidermal differentiation in the skin of K14-MMP10 mice (7).…”

Formalin-Fixed, Paraffin-Embedded Tissues (FFPE) as a Robust Source for the Profiling of Native and Protease-Generated Protein Amino Termini

“…6B). These results are in direct agreement with the prototypical profile of N-terminal acetylation and from previous studies on N-terminal acetylation in murine skin (35,48).…”

“…Acetylation is the most prevalent native N-terminal modification, which occurs in a posttranslational manner while Nterminal dimethylation is introduced during the TAILS procedure to protect free N termini that are often generated by endogenous proteolysis (14,35). The ratio of acetylated to dimethylated N termini differs to a limited extent between cryopreserved and FFPE specimens.…”