Cissé Moustapha Alfa, Sunyach Claire, Lefranc-Jullien Solveig, Postina Rolf, Vincent Bruno, Checler Frédéric
Institut de Pharmacologie Moléculaire et Cellulaire, du CNRS, UMR6097, Sophia-Antipolis, 06560 Valbonne, France.
J Biol Chem. 2005 Dec 9;280(49):40624-31. doi: 10.1074/jbc.M506069200. Epub 2005 Oct 18.
The cellular prion protein (PrP(c)) is physiologically cleaved in the middle of its 106-126 amino acid neurotoxic region at the 110/111 downward arrow112 peptidyl bond, yielding an N-terminal fragment referred to as N1. We recently demonstrated that two disintegrins, namely ADAM10 and ADAM17 (TACE, tumor necrosis factor alpha converting enzyme) participated in both constitutive and protein kinase C-regulated generation of N1, respectively. These proteolytic events were strikingly reminiscent of those involved in the so-called "alpha-secretase pathway" that leads to the production of secreted sAPPalpha from betaAPP. We show here, by transient and stable transfection analyses, that ADAM9 also participates in the constitutive secretion of N1 in HEK293 cells, TSM1 neurons, and mouse fibroblasts. Decreasing endogenous ADAM9 expression by an antisense approach drastically reduces both N1 and sAPPalpha recoveries. However, we establish that ADAM9 was unable to increase N1 and sAPPalpha productions after transient transfection in fibroblasts depleted of ADAM10. Accordingly, ADAM9 is unable to cleave a fluorimetric substrate of membrane-bound alpha-secretase activity in ADAM10(-/-) fibroblasts. However, we establish that co-expression of ADAM9 and ADAM10 in ADAM10-deficient fibroblasts leads to enhanced membrane-bound and released fluorimetric substrate hydrolyzing activity when compared with that observed after ADAM10 cDNA transfection alone in ADAM10(-/-) cells. Interestingly, we demonstrate that shedded ADAM10 displays the ability to cleave endogenous PrP(c) in fibroblasts. Altogether, these data provide evidence that ADAM9 is an important regulator of the physiological processing of PrP(c) and betaAPP but that this enzyme acts indirectly, likely by contributing to the shedding of ADAM10. ADAM9 could therefore represent, besides ADAM10, another potential therapeutic target to enhance the breakdown of the 106-126 and Abeta toxic domains of the prion and betaAPP proteins.
细胞朊蛋白（PrP(c)）在其106 - 126个氨基酸的神经毒性区域中部，于110/111↓112肽键处发生生理性切割，产生一个称为N1的N端片段。我们最近证明，两种解整合素，即ADAM10和ADAM17（肿瘤坏死因子α转换酶TACE）分别参与了N1的组成型和蛋白激酶C调节型生成。这些蛋白水解事件惊人地让人联想到参与所谓“α-分泌酶途径”的那些事件，该途径导致从β-淀粉样前体蛋白（betaAPP）产生分泌型sAPPα。我们在此通过瞬时和稳定转染分析表明，ADAM9也参与HEK293细胞、TSM1神经元和小鼠成纤维细胞中N1的组成型分泌。通过反义方法降低内源性ADAM9表达会大幅降低N1和sAPPα的回收率。然而，我们证实，在缺乏ADAM10的成纤维细胞中进行瞬时转染后，ADAM9无法增加N1和sAPPα的产生。因此，ADAM9无法在ADAM10(-/-)成纤维细胞中切割膜结合型α-分泌酶活性的荧光底物。然而，我们证实，与仅在ADAM10(-/-)细胞中转染ADAM10 cDNA后观察到的情况相比，在缺乏ADAM10的成纤维细胞中共表达ADAM9和ADAM10会导致膜结合型和释放型荧光底物水解活性增强。有趣的是，我们证明脱落的ADAM10具有在成纤维细胞中切割内源性PrP(c)的能力。总之这些数据表明，ADAM9是PrP(c)和betaAPP生理加工的重要调节因子，但该酶通过可能促进ADAM10的脱落而间接发挥作用。因此，除了ADAM10之外，ADAM9可能代表另一个潜在的治疗靶点，以增强朊蛋白和betaAPP蛋白106 - 126及Aβ毒性结构域的分解。
