γ-Secretase: Successive Tripeptide and Tetrapeptide Release from the Transmembrane Domain of β-Carboxyl Terminal Fragment
γ-Secretase: Successive Tripeptide and Tetrapeptide Release from the Transmembrane Domain of β-Carboxyl Terminal Fragment
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DOI：
10.1523/jneurosci.2362-09.2009
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发表时间：
2009-10-14
影响因子：
5.3
通讯作者：
Ihara, Yasuo
中科院分区：
文献类型：
--
作者：
Takami, Mako;Nagashima, Yu;Ihara, Yasuo
淀粉样β蛋白（A β）是一种与阿尔茨海默病相关的致病分子，由γ-分泌酶产生，其切割β-淀粉样前体蛋白在其跨膜结构域中间的β-羧基末端片段（β CTF）。细胞膜内的分裂是如何进行的一直是个谜。我们先前假设β CTF首先在膜-细胞质边界处裂解，产生两个长的A β，A β（48）和A β（49），其通过在每个步骤释放三个残基而进一步加工，分别产生A β（42）和A β（40）。为了验证这一假设，我们使用液相色谱串联质谱法（LC-MS/MS）来定量假定释放的特定三肽。使用CHAPSO（3-[（3-胆酰胺丙基）二甲基铵基]-2羟基-1-丙磺酸盐）-重建的γ-分泌酶系统，我们证实A β（49）通过连续释放两个或三个三肽转化为A β（43/40），A β（48）通过连续释放两个三肽或这些三肽加上另外的四肽转化为A β（42/38）。最出乎意料的是，LC-MS/MS定量揭示了肽生成中的诱导期，3-4分钟。外推时，每种三肽的每条时间线似乎与x轴上的相同点相交。基于连续反应动力学的数值模拟结果表明，诱导期是存在的。这些结果有力地表明，A β是通过γ-分泌酶逐步加工β CTF产生的。
Amyloid beta protein (A beta), a pathogenic molecule associated with Alzheimer's disease, is produced by gamma-secretase, which cleaves the beta-carboxyl terminal fragment (beta CTF) of beta-amyloid precursor protein in the middle of its transmembrane domain. How the cleavage proceeds within the membrane has long been enigmatic. We hypothesized previously that beta CTF is cleaved first at the membrane-cytoplasm boundary, producing two long A beta s, A beta(48) and A beta(49), which are processed further by releasing three residues at each step to produce A beta(42) and A beta(40), respectively. To test this hypothesis, we used liquid chromatography tandem mass spectrometry (LC-MS/MS) to quantify the specific tripeptides that are postulated to be released. Using CHAPSO (3-[(3-cholamidopropyl)dimethylammonio]-2hydroxyl-1-propanesulfonate)-reconstituted gamma-secretase system, we confirmed that A beta(49) is converted to A beta(43/40) by successively releasing two or three tripeptides and that A beta(48) is converted to A beta(42/38) by successively releasing two tripeptides or these plus an additional tetrapeptide. Most unexpectedly, LC-MS/MS quantification revealed an induction period, 3-4 min, in the generation of peptides. When extrapolated, each time line for each tripeptide appears to intercept the same point on the x-axis. According to numerical simulation based on the successive reaction kinetics, the induction period exists. These results strongly suggest that A beta is generated through the stepwise processing of beta CTF by gamma-secretase.