Transmembrane Substrate Determinants for γ-Secretase Processing of APP CTFβ.
Transmembrane Substrate Determinants for γ-Secretase Processing of APP CTFβ.
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DOI：
10.1021/acs.biochem.6b00718
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发表时间：
2016-10-11
期刊：
影响因子：
2.9
通讯作者：
Wolfe MS
中科院分区：
文献类型：
--
作者：
Fernandez MA;Biette KM;Dolios G;Seth D;Wang R;Wolfe MS
阿尔茨海默病（Alzheimer's disease，AD）的淀粉样β肽（amyloid β-peptide，Aβ）是由β淀粉样蛋白前体（amyloid β protein-precursor，APP CTFβ）的C-末端片段的跨膜区（transmembrane domain，TMD）内的γ-分泌酶复合物水解产生的。这种加工产生长度为38至49个残基的Aβ。有证据表明，Aβ肽谱是连续γ-分泌酶裂解的结果，首先在ε位点发生蛋白内切酶解，生成Aβ48或Aβ49，然后沿着两条产物线沿着每隔三个残基进行C末端修剪，生成较短的Aβ分泌形式：主要Aβ49-46-43-40线和次要Aβ48-45-42-38线。主要分泌的Aβ种类是Aβ40和Aβ42，与Aβ40相比，较长的、易于聚集的Aβ42的比例增加被广泛认为在AD发病机制中很重要。我们研究了ε位点蛋白内切酶和γ-分泌酶对CTFβ的羧肽酶修饰的特异性和效率的TMD底物决定因素。我们确定，中间体Aβ49的C端负电荷在γ-分泌酶对其的修剪中不起作用。拟肽探针表明γ-分泌酶具有S1 '、S2'和S3'口袋，通过这些口袋可以确定三肽的修剪。然而，ε位点周围残基的缺失表明TMD内三个残基的深度不是CTFβ内切蛋白水解ε切割位置的决定因素。我们还发现，CTFβ TMD螺旋在ε位点附近的不稳定性显著增加了内蛋白水解，并且羧肽酶切割位点附近的螺旋不稳定性促进了γ-分泌酶对C-末端的修剪。此外，我们发现CTFβ二聚体不被γ-分泌酶内切蛋白水解。这些结果支持了这样一种模型，其中沿着底物的未二聚化的单螺旋TMD的残基沿着阵列的初始相互作用决定了初始ε切割的位点，并且螺旋解旋对于内蛋白水解和羧肽酶修剪都是必需的。
The amyloid β-peptide (Aβ) of Alzheimer’s disease (AD) is generated by proteolysis within the transmembrane domain (TMD) of a C-terminal fragment of the amyloid β protein-precursor (APP CTFβ) by the γ-secretase complex. This processing produces Aβ ranging from 38 to 49 residues in length. Evidence suggests that this spectrum of Aβ peptides is the result of successive γ-secretase cleavages, with endoproteolysis first occurring at the ε sites to generate Aβ48 or Aβ49, followed by C-terminal trimming mostly every three residues along two product lines to generate shorter, secreted forms of Aβ: the primary Aβ49-46-43-40 line and a minor Aβ48-45-42-38 line. The major secreted Aβ species are Aβ40 and Aβ42, and an increased proportion of the longer, aggregation-prone Aβ42 compared to Aβ40 is widely thought to be important in AD pathogenesis. We examined TMD substrate determinants of the specificity and efficiency of ε site endoproteolysis and carboxypeptidase trimming of CTFβ by γ-secretase. We determined that the C-terminal negative charge of the intermediate Aβ49 does not play a role in its trimming by γ-secretase. Peptidomimetic probes suggest that γ-secretase has S1’, S2’, and S3’ pockets, through which trimming by tripeptides may be determined. However, deletion of residues around the ε sites demonstrates that a depth of three residues within the TMD is not a determinant of the location of endoproteolytic ε cleavage of CTFβ. We also show that instability of the CTFβ TMD helix near the ε site significantly increases endoproteolysis, and that helical instability near the carboxypeptidase cleavage sites facilitates C-terminal trimming by γ-secretase. In addition, we found that CTFβ dimers are not endoproteolyzed by γ-secretase. These results support a model in which initial interaction of the array of residues along the undimerized single helical TMD of substrates dictates the site of initial ε cleavage and that helix unwinding is essential for both endoproteolysis and carboxypeptidase trimming.
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