Identification of BACE1 cleavage sites in human voltage-gated sodium channel beta 2 subunit.
Identification of BACE1 cleavage sites in human voltage-gated sodium channel beta 2 subunit.
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DOI：
10.1186/1750-1326-5-61
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发表时间：
2010-12-23
影响因子：
15.1
通讯作者：
Kovacs DM
中科院分区：
文献类型：
--
作者：
Gersbacher MT;Kim DY;Bhattacharyya R;Kovacs DM
电压门控钠通道β2亚基（Navβ2）是BACE 1（β-site APP cleaving enzyme）和γ-secretase（γ-secretase）的生理底物，这两种蛋白水解酶是阿尔茨海默病发病机制的核心。在此之前，我们已经发现BACE 1和γ-分泌酶对Navβ2的加工调节神经细胞中的钠通道代谢。在本研究中，我们鉴定了人Navβ2中的BACE 1切割位点。我们使用无细胞BACE 1切割试验和质谱法在人Navβ2的细胞外结构域中发现了一个主要（147-148 L↓M，其中↓表示切割位点）和一个次要（144145 L↓Q）BACE 1切割位点。接下来，我们将两个不同的双突变引入人Navβ2中鉴定的主要BACE 1切割位点：147 LM/VI和147 LM/AA。在无细胞BACE 1裂解试验中，两种突变均显著降低内源性BACE 1对人Navβ2的裂解。这两种突变均不影响Navβ2的亚细胞定位，这一点通过共聚焦荧光显微镜和富含胆固醇结构域的亚细胞分级分离证实。最后，野生型和突变的Navβ2在B104大鼠神经母细胞瘤细胞中沿着BACE 1表达沿着。尽管α-分泌酶仍然活跃地裂解突变蛋白，但与表达野生型Navβ2的细胞相比，Navβ2裂解产物在表达Nav β 2的细胞中（147 LM/VI）减少约50%，在表达Nav β 2的细胞中（147 LM/AA）减少约75%。我们在人Navβ2中鉴定了一个主要（147-148 L↓M）和一个次要（144-145 L↓Q）BACE 1切割位点。我们的体外和基于细胞的结果清楚地表明，147-148 L↓M是人Navβ2中的主要BACE 1切割位点。这些发现扩展了我们对BACE 1在电压门控钠通道代谢中作用的理解。
The voltage-gated sodium channel β2 subunit (Navβ2) is a physiological substrate of BACE1 (β-site APP cleaving enzyme) and γ-secretase, two proteolytic enzymes central to Alzheimer's disease pathogenesis. Previously, we have found that the processing of Navβ2 by BACE1 and γ-secretase regulates sodium channel metabolism in neuronal cells. In the current study we identified the BACE1 cleavage sites in human Navβ2. We found a major (147-148 L↓M, where ↓ indicates the cleavage site) and a minor (144145 L↓Q) BACE1 cleavage site in the extracellular domain of human Navβ2 using a cell-free BACE1 cleavage assay followed by mass spectrometry. Next, we introduced two different double mutations into the identified major BACE1 cleavage site in human Navβ2: 147LM/VI and 147LM/AA. Both mutations dramatically decreased the cleavage of human Navβ2 by endogenous BACE1 in cell-free BACE1 cleavage assays. Neither of the two mutations affected subcellular localization of Navβ2 as confirmed by confocal fluorescence microscopy and subcellular fractionation of cholesterol-rich domains. Finally, wildtype and mutated Navβ2 were expressed along BACE1 in B104 rat neuroblastoma cells. In spite of α-secretase still actively cleaving the mutant proteins, Navβ2 cleavage products decreased by ~50% in cells expressing Navβ2 (147LM/VI) and ~75% in cells expressing Navβ2 (147LM/AA) as compared to cells expressing wildtype Navβ2. We identified a major (147-148 L↓M) and a minor (144-145 L↓Q) BACE1 cleavage site in human Navβ2. Our in vitro and cell-based results clearly show that the 147-148 L↓M is the major BACE1 cleavage site in human Navβ2. These findings expand our understanding of the role of BACE1 in voltage-gated sodium channel metabolism.
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DOI：
10.1016/s0006-291x(02)02635-9
发表时间：
2002-12-06
影响因子：
3.1
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