Institute of Biochemistry, Christian-Albrechts-University, 24118, Kiel, Germany.
Cell Mol Life Sci. 2013 Jan;70(2):309-33. doi: 10.1007/s00018-012-1106-2. Epub 2012 Sep 1.
The in vivo roles of meprin metalloproteases in pathophysiological conditions remain elusive. Substrates define protease roles. Therefore, to identify natural substrates for human meprin α and β we employed TAILS (terminal amine isotopic labeling of substrates), a proteomics approach that enriches for N-terminal peptides of proteins and cleavage fragments. Of the 151 new extracellular substrates we identified, it was notable that ADAM10 (a disintegrin and metalloprotease domain-containing protein 10)-the constitutive α-secretase-is activated by meprin β through cleavage of the propeptide. To validate this cleavage event, we expressed recombinant proADAM10 and after preincubation with meprin β, this resulted in significantly elevated ADAM10 activity. Cellular expression in murine primary fibroblasts confirmed activation. Other novel substrates including extracellular matrix proteins, growth factors and inhibitors were validated by western analyses and enzyme activity assays with Edman sequencing confirming the exact cleavage sites identified by TAILS. Cleavages in vivo were confirmed by comparing wild-type and meprin(-/-) mice. Our finding of cystatin C, elafin and fetuin-A as substrates and natural inhibitors for meprins reveal new mechanisms in the regulation of protease activity important for understanding pathophysiological processes.
在生理病理条件下，金属蛋白酶 meprin 的体内作用仍难以捉摸。底物决定了蛋白酶的作用。因此，为了鉴定人源 meprin α 和 β 的天然底物，我们采用了 TAILS（末端胺同位素标记的底物），这是一种可富集蛋白质 N 端肽和切割片段的蛋白质组学方法。在我们鉴定的 151 种新的细胞外底物中，值得注意的是，ADAM10（含有去整合素和金属蛋白酶结构域的蛋白 10）-组成型 α 分泌酶-通过 meprin β 对前肽的切割而被激活。为了验证这一切割事件，我们表达了重组的 proADAM10，并且在与 meprin β 预孵育后，这导致 ADAM10 活性显著升高。在鼠原代成纤维细胞中的细胞表达证实了激活。其他新的底物，包括细胞外基质蛋白、生长因子和抑制剂，通过 Western 分析和 Edman 测序的酶活性测定进行了验证，Edman 测序确认了 TAILS 鉴定的精确切割位点。通过比较野生型和 meprin(-/-)小鼠，证实了体内的切割。我们发现半胱氨酸蛋白酶抑制剂 C、elafin 和胎球蛋白 A 是 meprin 的底物和天然抑制剂，这揭示了调节蛋白酶活性的新机制，对理解生理病理过程很重要。
