MMP10 degradomics in keratinocytes

Matrix metalloproteinases (MMPs) are important players in skin homeostasis, wound repair, and in the pathogenesis of skin cancer. One member – MMP10 – is specifically expressed in wound keratinocytes and in epithelial cancer cells, but its function is unclear and epidermal substrates are poorly characterized. To unravel the role of MMP10 in the skin, we employed Terminal Amine Isotopic Labeling of Substrates (TAILS), an iTRAQ-based quantitative proteomics technique for the discovery of protease substrates and their cleavage sites, to monitor MMP10-dependent proteolysis over time in secretomes from keratinocytes. By time-resolved abundance clustering of neo-N termini we revealed a cleavage site specificity preference of MMP10 for glutamate in the P1 position and identified the α6 integrin subunit, cysteine-rich angiogenic inducer 61 and dermokine as novel MMP10 substrates. Moreover, we confirmed the same MMP10-dependent processing of dermokine in vivo by TAILS analysis of epidermis from transgenic mice that overexpress a constitutively active mutant of MMP10 in basal keratinocytes. Since these substrates have been associated with cell adhesion, differentiation and senescence, MMP10 might contribute to modulation of these processes during skin repair.

基质金属蛋白酶（Matrix metalloproteinases, MMPs）是参与皮肤稳态、伤口修复及皮肤癌发病机制的重要调控因子。其中一员——基质金属蛋白酶10（MMP10）——可特异性表达于伤口角质形成细胞与上皮癌细胞中，但其具体功能尚未明确，表皮相关底物的特征也未被充分解析。为阐明MMP10在皮肤中的作用，本研究采用了底物末端胺同位素标记（Terminal Amine Isotopic Labeling of Substrates, TAILS）技术：该技术是一种基于iTRAQ的定量蛋白质组学方法，用于挖掘蛋白酶底物及其切割位点，借此对角质形成细胞分泌组中MMP10介导的蛋白水解过程进行时序监测。通过对新N末端进行时间分辨率的丰度聚类分析，我们揭示了MMP10在P1位偏好谷氨酸的切割位点特异性，并鉴定出α6整合素亚基、富半胱氨酸血管生成诱导因子61（cysteine-rich angiogenic inducer 61）以及真皮调蛋白（dermokine）作为新型MMP10底物。此外，我们通过对在基底角质形成细胞中过表达组成型激活MMP10突变体的转基因小鼠表皮开展TAILS分析，在体内验证了MMP10对真皮调蛋白的上述切割加工过程。鉴于上述底物均与细胞黏附、细胞分化及细胞衰老相关，MMP10可能在皮肤修复过程中参与调控上述生物学进程。