Effects of neprilysin chimeric proteins targeted to subcellular compartments on amyloid β peptide clearance in primary neurons
Effects of neprilysin chimeric proteins targeted to subcellular compartments on amyloid β peptide clearance in primary neurons
复制标题
DOI：
10.1074/jbc.m401891200
复制
发表时间：
2004-07-16
影响因子：
4.8
通讯作者：
Saido, TC
中科院分区：
文献类型：
--
作者：
Hama, E;Shirotani, K;Saido, TC
脑啡肽酶 (NEP) 是大脑中一种限速淀粉样β肽 (Abeta) 降解酶。我们之前证明，原代皮质神经元中脑啡肽酶的过度表达不仅显着降低细胞外，而且还显着降低细胞内 Abeta 水平。为了研究脑啡肽酶最有效地降解 Abeta 的亚细胞区室，我们在神经元中表达了含有各种亚细胞区室靶向结构域的脑啡肽酶嵌合蛋白。 Sec12-NEP、β-半乳糖苷 α2,6-唾液酸转移酶-NEP、转铁蛋白受体-NEP 和生长相关蛋白 43-NEP 分别成功分选至内质网、反式高尔基体网络、早期/再循环内体和脂筏。我们发现在细胞内，野生型脑啡肽酶和所有嵌合体都表现出相同的 Abeta40 降解活性。 Abeta40 比 Abeta42 被更有效地清除，并且细胞内 Abeta 的这种趋势比细胞外 Abeta 更大。野生型和跨高尔基体网络靶向的 ST-NEP 比其他嵌合体清除更多的细胞内 Abeta42。野生型脑啡肽酶比任何嵌合体都更有效地清除细胞外 Abeta，其中内质网靶向的 Sec12-NEP 的效果最差。这些观察结果表明，不同的细胞内区室可能参与不同 Abeta 池（Abeta40 和 Abeta42）的代谢，以在细胞内保留或循环并分泌到细胞外，并且野生型脑啡肽酶中的内源靶向信号针对 Abeta 的整体神经元清除进行了很好的优化。
Neprilysin (NEP) is a rate-limiting amyloid beta peptide (Abeta)-degrading enzyme in the brain. We demonstrated previously that overexpression of neprilysin in primary cortical neurons remarkably decreased not only extracellular but also intracellular Abeta levels. To investigate the subcellular compartments where neprilysin degrades Abeta most efficiently, we expressed neprilysin chimeric proteins containing various subcellular compartment-targeting domains in neurons. Sec12-NEP, beta-galactoside alpha2,6-sialyltransferase-NEP, transferrin receptor-NEP, and growth-associated protein 43-NEP were successfully sorted to the endoplasmic reticulum, trans-Golgi network, early/recycling endosomes, and lipid rafts, respectively. We found that intracellularly, wild-type neprilysin and all the chimeras showed equivalent Abeta40-degrading activities. Abeta40 was more effectively cleared than Abeta42, and this tendency was greater for intracellular Abeta than for extracellular Abeta. Wild-type and trans-Golgi network- targeted ST-NEP cleared more intracellular Abeta42 than the other chimeras. Wild-type neprilysin cleared extracellular Abeta more effectively than any of the chimeras, among which endoplasmic reticulum-targeted Sec12-NEP was the least effective. These observations indicate that different intracellular compartments may be involved in the metabolism of distinct pools of Abeta (Abeta40 and Abeta42) to be retained or recycled intracellularly and to be secreted extracellularly, and that the endogenous targeting signal in wildtype neprilysin is well optimized for the overall neuronal clearance of Abeta.