Holmes Oliver, Paturi Swetha, Wolfe Michael S, Selkoe Dennis J
Center for Neurologic Diseases, Brigham and Women's Hospital, Boston, MA, USA; Harvard Medical School, Boston, MA, USA.
J Neurochem. 2014 Oct;131(1):94-100. doi: 10.1111/jnc.12772. Epub 2014 Jun 18.
The 19-transmembrane, multisubunit γ-secretase complex generates the amyloid β-peptide (Aβ) of Alzheimer's disease (AD) by an unusual intramembrane proteolysis of the β-amyloid precursor protein. The complex, which similarly processes many other type 1 transmembrane substrates, is composed of presenilin, Aph1, nicastrin, and presenilin enhancer (Pen-2), all of which are necessary for proper complex maturation and enzymatic activity. Obtaining a high-resolution atomic structure of the intact complex would greatly aid the rational design of compounds to modulate activity but is a very difficult task. A complementary method is to generate structures for each individual subunit to allow one to build a model of the entire complex. Here, we describe a method by which recombinant human Pen-2 can be purified from bacteria to > 95% purity at milligram quantities per liter, utilizing a maltose binding protein tag to both increase solubility and facilitate purification. Expressing the same construct in mammalian cells, we show that the large N-terminal maltose binding protein tag on Pen-2 still permits incorporation into the complex and subsequent presenilin-1 endoproteolysis, nicastrin glycosylation and proteolytic activity. These new methods provide valuable tools to study the structure and function of Pen-2 and the γ-secretase complex.
19跨膜多亚基γ-分泌酶复合物通过对β-淀粉样前体蛋白进行异常的膜内蛋白水解作用，产生阿尔茨海默病（AD）的淀粉样β肽（Aβ）。该复合物同样可处理许多其他1型跨膜底物，它由早老素、Aph1、尼卡斯特林和早老素增强子（Pen-2）组成，所有这些对于复合物的正常成熟和酶活性都是必需的。获得完整复合物的高分辨率原子结构将极大地有助于合理设计调节活性的化合物，但这是一项非常艰巨的任务。一种补充方法是为每个单独的亚基生成结构，以便构建整个复合物的模型。在这里，我们描述了一种方法，利用麦芽糖结合蛋白标签来增加溶解度并促进纯化，从而可以从细菌中以每升毫克量的规模纯化重组人Pen-2，纯度超过95%。在哺乳动物细胞中表达相同的构建体时，我们发现Pen-2上的大N端麦芽糖结合蛋白标签仍允许其掺入复合物并随后进行早老素-1的内切蛋白水解、尼卡斯特林的糖基化和蛋白水解活性。这些新方法为研究Pen-2和γ-分泌酶复合物的结构与功能提供了有价值的工具。
