From the Departments of Neuroscience.
Protein Chemistry.
J Biol Chem. 2014 Nov 7;289(45):30990-1000. doi: 10.1074/jbc.M114.589069. Epub 2014 Sep 24.
Pathogenic mutations in the amyloid precursor protein (APP) gene have been described as causing early onset familial Alzheimer disease (AD). We recently identified a rare APP variant encoding an alanine-to-threonine substitution at residue 673 (A673T) that confers protection against development of AD (Jonsson, T., Atwal, J. K., Steinberg, S., Snaedal, J., Jonsson, P. V., Bjornsson, S., Stefansson, H., Sulem, P., Gudbjartsson, D., Maloney, J., Hoyte, K., Gustafson, A., Liu, Y., Lu, Y., Bhangale, T., Graham, R. R., Huttenlocher, J., Bjornsdottir, G., Andreassen, O. A., Jönsson, E. G., Palotie, A., Behrens, T. W., Magnusson, O. T., Kong, A., Thorsteinsdottir, U., Watts, R. J., and Stefansson, K. (2012) Nature 488, 96-99). The Ala-673 residue lies within the β-secretase recognition sequence and is part of the amyloid-β (Aβ) peptide cleavage product (position 2 of Aβ). We previously demonstrated that the A673T substitution makes APP a less favorable substrate for cleavage by BACE1. In follow-up studies, we confirm that A673T APP shows reduced cleavage by BACE1 in transfected mouse primary neurons and in isogenic human induced pluripotent stem cell-derived neurons. Using a biochemical approach, we show that the A673T substitution modulates the catalytic turnover rate (V(max)) of APP by the BACE1 enzyme, without affecting the affinity (K(m)) of the APP substrate for BACE1. We also show a reduced level of Aβ(1-42) aggregation with A2T Aβ peptides, an observation not conserved in Aβ(1-40) peptides. When combined in a ratio of 1:9 Aβ(1-42)/Aβ(1-40) to mimic physiologically relevant mixtures, A2T retains a trend toward slowed aggregation kinetics. Microglial uptake of the mutant Aβ(1-42) peptides correlated with their aggregation level. Cytotoxicity of the mutant Aβ peptides was not dramatically altered. Taken together, our findings demonstrate that A673T, a protective allele of APP, reproducibly reduces amyloidogenic processing of APP and also mildly decreases Aβ aggregation. These effects could together have an additive or even synergistic impact on the risk of developing AD.
淀粉样前体蛋白（APP）基因中的致病突变已被描述为导致早发性家族性阿尔茨海默病（AD）。我们最近发现了一种罕见的 APP 变体，其编码的丙氨酸到苏氨酸取代位于残基 673（A673T），该变体可防止 AD 的发展（Jonsson，T.，Atwal，J. K.，Steinberg，S.，Snaedal，J.，Jonsson，P. V.，Bjornsson，S.，Stefansson，H.，Sulem，P.，Gudbjartsson，D.，Maloney，J.，Hoyte，K.，Gustafson，A.，Liu，Y.，Lu，Y.，Bhangale，T.，Graham，R. R.，Huttenlocher，J.，Bjornsdottir，G.，Andreassen，O. A.，Jönsson，E. G.，Palotie，A.，Behrens，T. W.，Magnusson，O. T.，Kong，A.，Thorsteinsdottir，U.，Watts，R. J.，and Stefansson，K.（2012）Nature 488, 96-99）。Ala-673 残基位于 β-分泌酶识别序列内，是淀粉样-β（Aβ）肽裂解产物的一部分（Aβ的位置 2）。我们之前证明，A673T 取代使 APP 成为 BACE1 切割的不太有利的底物。在后续研究中，我们证实 A673T APP 在转染的小鼠原代神经元和同种异体人诱导多能干细胞衍生的神经元中显示出 BACE1 切割减少。使用生化方法，我们表明 A673T 取代调节 BACE1 酶对 APP 的催化周转率（Vmax），而不影响 APP 底物与 BACE1 的亲和力（K m）。我们还观察到 A2T Aβ 肽的 Aβ（1-42）聚集水平降低，而 Aβ（1-40）肽则没有观察到这种情况。当以模拟生理相关混合物的 1:9 Aβ（1-42）/Aβ（1-40）的比例组合时，A2T 仍保持聚集动力学减慢的趋势。突变 Aβ（1-42）肽的小胶质细胞摄取与它们的聚集水平相关。突变 Aβ 肽的细胞毒性没有明显改变。总之，我们的发现表明，APP 的保护性等位基因 A673T 可重复降低 APP 的淀粉样蛋白形成加工，并且还轻度降低 Aβ 聚集。这些影响可能共同对 AD 发病风险产生相加甚至协同作用。
