Institute of Health and Biomedical Innovation, Queensland University of Technology, Brisbane, Queensland, Australia; Laboratory of Genetic Skin Diseases, INSERM UMR 1163 and Imagine Institute of Genetic Diseases, Paris, France; Université Paris V Descartes-Sorbonne Paris Cité, Paris, France.
Laboratory of Genetic Skin Diseases, INSERM UMR 1163 and Imagine Institute of Genetic Diseases, Paris, France; Université Paris V Descartes-Sorbonne Paris Cité, Paris, France.
J Invest Dermatol. 2017 Feb;137(2):430-439. doi: 10.1016/j.jid.2016.09.017. Epub 2016 Sep 30.
Proteases have pivotal roles in the skin's outermost layer, the epidermis. In the stratum corneum, serine proteases from the kallikrein-related peptidase (KLK) family have been implicated in several key homeostatic processes, including desquamation. However, the precise contribution of specific KLKs to each process remains unclear. To address this, we used a chemical biology approach and designed selective substrates and inhibitors for KLK7, the most abundant KLK protease in the stratum corneum. The resulting KLK7 inhibitor is the most potent inhibitor of this protease reported to date (K = 140 pM), and displays at least 1,000-fold selectivity over several proteases that are related by function (KLK5 and KLK14) or specificity (chymotrypsin). We then used substrates and inhibitors for KLK5, KLK7, and KLK14 to explore the activity of each protease in the stratum corneum using casein zymography and an ex vivo desquamation assay. These experiments provide the most detailed assessment of each KLK's contribution to corneocyte shedding in the plantar stratum corneum, revealing that inhibition of KLK7 alone is sufficient to block shedding, whereas KLK5 is also a major contributor. Collectively, these findings unveil chemical tools for studying KLK activity and demonstrate their potential for characterizing KLK biological functions in epidermal homeostasis.
蛋白酶在皮肤的最外层——表皮中起着关键作用。在角质层中，来自激肽释放酶相关肽酶（KLK）家族的丝氨酸蛋白酶被认为参与了几个关键的动态平衡过程，包括脱屑。然而，特定 KLK 对每个过程的确切贡献仍不清楚。为了解决这个问题，我们使用了一种化学生物学方法，为 KLK7 设计了选择性的底物和抑制剂，KLK7 是角质层中最丰富的 KLK 蛋白酶。由此产生的 KLK7 抑制剂是迄今为止报道的这种蛋白酶最有效的抑制剂（K=140 pM），对功能相关（KLK5 和 KLK14）或特异性（糜蛋白酶）的几种蛋白酶具有至少 1000 倍的选择性。然后，我们使用 KLK5、KLK7 和 KLK14 的底物和抑制剂，通过酪蛋白酶谱法和体外脱屑测定法，在角质层中探索每种蛋白酶的活性。这些实验提供了对每种 KLK 在足底角质层角质细胞脱落中的贡献的最详细评估，表明单独抑制 KLK7 足以阻止脱落，而 KLK5 也是主要贡献者。总的来说，这些发现揭示了用于研究 KLK 活性的化学工具，并展示了它们在表皮动态平衡中表征 KLK 生物学功能的潜力。
