Malopolska Centre of Biotechnology, Jagiellonian University, 30-387 Krakow, Poland.
Faculty of Biochemistry, Biophysics and Biotechnology, Jagiellonian University, 30-387 Krakow, Poland.
Int J Mol Sci. 2020 Jun 19;21(12):4383. doi: 10.3390/ijms21124383.
Kallikrein-related peptidases (KLKs) and matrix metalloproteinases (MMPs) are secretory proteinases known to proteolytically process components of the extracellular matrix, modulating the pericellular environment in physiology and in pathologies. The interconnection between these families remains elusive. To assess the cross-activation of these families, we developed a peptide, fusion protein-based exposition system (age of posed amino acid sequences, CleavEx) aiming at investigating the potential of KLK14 to recognize and hydrolyze proMMP sequences. Initial assessment identified ten MMP activation domain sequences which were validated by Edman degradation. The analysis revealed that membrane-type MMPs (MT-MMPs) are targeted by KLK14 for activation. Correspondingly, proMMP14-17 were investigated in vitro and found to be effectively processed by KLK14. Again, the expected neo-N-termini of the activated MT-MMPs was confirmed by Edman degradation. The effectiveness of proMMP activation was analyzed by gelatin zymography, confirming the release of fully active, mature MT-MMPs upon KLK14 treatment. Lastly, MMP14 was shown to be processed on the cell surface by KLK14 using murine fibroblasts overexpressing human MMP14. Herein, we propose KLK14-mediated selective activation of cell-membrane located MT-MMPs as an additional layer of their regulation. As both, KLKs and MT-MMPs, are implicated in cancer, their cross-activation may constitute an important factor in tumor progression and metastasis.
激肽释放酶相关肽酶（KLKs）和基质金属蛋白酶（MMPs）是分泌性蛋白水解酶，已知可对细胞外基质的成分进行蛋白水解加工，调节生理和病理状态下细胞周围环境。这两个家族之间的联系仍然难以捉摸。为了评估这两个家族之间的交叉激活，我们开发了一种基于肽和融合蛋白的暴露系统（CleavEx），旨在研究 KLK14 识别和水解 proMMP 序列的潜力。初步评估确定了十个 MMP 激活结构域序列，并用 Edman 降解进行了验证。分析表明，KLK14 靶向膜型 MMPs（MT-MMPs）进行激活。相应地，研究了体外的 proMMP14-17，并发现它们可被 KLK14 有效加工。同样，通过 Edman 降解证实了预期的激活 MT-MMP 的新 N-末端。通过明胶酶谱分析分析了 proMMP 激活的效果，证实 KLK14 处理后可释放完全活性的成熟 MT-MMPs。最后，使用过表达人 MMP14 的小鼠成纤维细胞，证明 KLK14 在细胞表面对 MMP14 进行加工。在此，我们提出 KLK14 介导的细胞表面定位的 MT-MMPs 的选择性激活作为其调节的附加层。由于 KLKs 和 MT-MMPs 都与癌症有关，它们的交叉激活可能是肿瘤进展和转移的重要因素。
