Cissé Moustapha Alfa, Gandreuil Céline, Hernandez Jean-François, Martinez Jean, Checler Frédéric, Vincent Bruno
Institut de Pharmacologie Moléculaire et Cellulaire, UMR6097 CNRS/UNSA, Equipe labélisée Fondation pour la Recherche Médicale, Valbonne, France.
Biochem Biophys Res Commun. 2006 Aug 18;347(1):254-60. doi: 10.1016/j.bbrc.2006.06.065. Epub 2006 Jun 21.
Under normal conditions, the cellular prion protein (PrP(c)) undergoes a proteolytic attack between amino acids 111 and 112 which gives rise to the N-terminal secreted N1 fragment and its C-terminal membrane-tethered counterpart C1. Importantly, this cleavage precludes the integrity of the neurotoxic 106-126 sequence. Here, we describe an original and reliable assay based on a quenched fluorimetric substrate (JMV2770) encompassing the 111/112 sequence of PrP(c). In whole brain homogenate, the JMV2770-hydrolysing activity is optimal at neutral pH and sensitive to the metalloprotease inhibitor BB3103 but not to acidic and serine protease blockers. JMV2770 is efficiently cleaved by intact HEK293 cells and fibroblasts in culture, consistent with an hydrolysis by a typical ectoprotease. Overexpressions of alpha-secretases a disintegrin and metalloprotease-9 (ADAM9), ADAM10 or TACE (ADAM17) in human cells increase BB3103-sensitive JMV2770 hydrolysis, while invalidation of ADAM10 and TACE or reduced expression of ADAM9 by an antisense approach significantly reduced its cleavage. Finally, analysis of JMV2770 hydrolysis following transient transfection of ADAM10 or ADAM9 cDNA in ADAM10(-/-) fibroblasts allowed to confirm our previous data establishing that ADAM9 does not behave as a genuine alpha-secretase but rather acts as an important upstream regulator of ADAM10 activity.
在正常情况下，细胞朊蛋白（PrP(c)）在氨基酸111和112之间受到蛋白水解攻击，产生N端分泌的N1片段及其C端膜结合对应物C1。重要的是，这种切割排除了神经毒性106 - 126序列的完整性。在此，我们描述了一种基于包含PrP(c) 111/112序列的淬灭荧光底物（JMV2770）的原始且可靠的检测方法。在全脑匀浆中，JMV2770水解活性在中性pH下最佳，对金属蛋白酶抑制剂BB3103敏感，但对酸性和丝氨酸蛋白酶阻滞剂不敏感。JMV2770在培养的完整HEK293细胞和成纤维细胞中被有效切割，这与典型的外切蛋白酶水解一致。在人细胞中过表达α-分泌酶解整合素金属蛋白酶-9（ADAM9）、ADAM10或肿瘤坏死因子α转换酶（TACE，ADAM17）会增加对BB3103敏感的JMV2770水解，而通过反义方法使ADAM10和TACE失活或降低ADAM9的表达会显著降低其切割。最后，在ADAM10(-/-)成纤维细胞中瞬时转染ADAM10或ADAM9 cDNA后对JMV2770水解的分析，证实了我们之前的数据，即ADAM9并非真正的α-分泌酶，而是作为ADAM10活性的重要上游调节因子发挥作用。
