Bekhouche Mourad, Leduc Cedric, Dupont Laura, Janssen Lauriane, Delolme Frederic, Vadon-Le Goff Sandrine, Smargiasso Nicolas, Baiwir Dominique, Mazzucchelli Gabriel, Zanella-Cleon Isabelle, Dubail Johanne, De Pauw Edwin, Nusgens Betty, Hulmes David J S, Moali Catherine, Colige Alain
Laboratory of Connective Tissues Biology, University of Liège, Liège, Belgium;
Tissue Biology and Therapeutic Engineering, Centre National de la Recherche Scientifique/University of Lyon Unité Mixte de Recherche 5305, Lyon, France; and Protein Science Facility, Institute for the Biology and Chemistry of Proteins, Unité Mixte de Service 3444, Lyon, France.
FASEB J. 2016 May;30(5):1741-56. doi: 10.1096/fj.15-279869. Epub 2016 Jan 6.
A disintegrin and metalloproteinase with thrombospondin type I motif (ADAMTS)2, 3, and 14 are collectively named procollagen N-proteinases (pNPs) because of their specific ability to cleave the aminopropeptide of fibrillar procollagens. Several reports also indicate that they could be involved in other biological processes, such as blood coagulation, development, and male fertility, but the potential substrates associated with these activities remain unknown. Using the recently described N-terminal amine isotopic labeling of substrate approach, we analyzed the secretomes of human fibroblasts and identified 8, 17, and 22 candidate substrates for ADAMTS2, 3, and 14, respectively. Among these newly identified substrates, many are components of the extracellular matrix and/or proteins related to cell signaling such as latent TGF-β binding protein 1, TGF-β RIII, and dickkopf-related protein 3. Candidate substrates for the 3 ADAMTS have been biochemically validated in different contexts, and the implication of ADAMTS2 in the control of TGF-β activity has been further demonstrated in human fibroblasts. Finally, the cleavage site specificity was assessed showing a clear and unique preference for nonpolar or slightly hydrophobic amino acids. This work shows that the activities of the pNPs extend far beyond the classically reported processing of the aminopropeptide of fibrillar collagens and that they should now be considered as multilevel regulators of matrix deposition and remodeling.-Bekhouche, M., Leduc, C., Dupont, L., Janssen, L., Delolme, F., Vadon-Le Goff, S., Smargiasso, N., Baiwir, D., Mazzucchelli, G., Zanella-Cleon, I., Dubail, J., De Pauw, E., Nusgens, B., Hulmes, D. J. S., Moali, C., Colige, A. Determination of the substrate repertoire of ADAMTS2, 3, and 14 significantly broadens their functions and identifies extracellular matrix organization and TGF-β signaling as primary targets.
具有I型血小板反应蛋白基序的解整合素和金属蛋白酶（ADAMTS）2、3和14由于其能够特异性切割原纤维状前胶原的氨基端前肽，因而统称为前胶原N蛋白酶（pNP）。一些报告还指出，它们可能参与其他生物学过程，如血液凝固、发育和男性生育，但与这些活性相关的潜在底物仍不清楚。利用最近描述的底物N端胺同位素标记方法，我们分析了人成纤维细胞的分泌蛋白质组，分别鉴定出ADAMTS2、3和14的8种、17种和22种候选底物。在这些新鉴定的底物中，许多是细胞外基质的成分和/或与细胞信号传导相关的蛋白质，如潜伏性转化生长因子-β结合蛋白1、转化生长因子-βRIII和Dickkopf相关蛋白3。这三种ADAMTS的候选底物已在不同情况下得到生物化学验证，并且ADAMTS2在人成纤维细胞中对转化生长因子-β活性的调控作用得到了进一步证明。最后，对切割位点特异性进行了评估，结果显示对非极性或轻度疏水氨基酸有明显且独特的偏好。这项研究表明，pNP的活性远远超出了经典报道的对原纤维状胶原氨基端前肽的加工，现在应将它们视为基质沉积和重塑的多级调节因子。-贝库切，M.，勒迪克，C.，杜邦，L.，扬森，L.，德洛尔姆，F.，瓦东-勒戈夫，S.，斯马尔贾索，N.，贝维尔，D.，马祖切利，G.，扎内拉-克莱昂，I.，迪拜尔，J.，德保，E.，努斯根斯，B.，赫尔姆斯，D.J.S.，莫阿利，C.，科利热，A.确定ADAMTS2、3和14的底物谱显著拓宽了它们的功能，并将细胞外基质组织和转化生长因子-β信号传导确定为主要靶点。
