Tomita T
Laboratory of Neuropathology and Neuroscience, Graduate School of Pharmaceutical Sciences, The University of Tokyo, Tokyo, Japan.
Methods Enzymol. 2017;584:185-205. doi: 10.1016/bs.mie.2016.10.033. Epub 2016 Nov 23.
Presenilin is a catalytic subunit of γ-secretase, which hydrolyzes several transmembrane proteins within the lipid bilayer, together with binding cofactors such as nicastrin, Aph-1, and Pen-2. However, the structural basis as well as molecular mechanism of this unusual proteolytic process remains unknown. We have analyzed the structure and function relationships of presenilin using the substituted-cysteine accessibility method (SCAM), which enables identification of the hydrophilic environment by the accessibility of sulfhydryl reagents to cysteine residues introduced at a desired position. In combination with small molecule inhibitors/modulators and cross-linking experiments, we were able to identify certain residues and regions of presenilin that contribute to its intramembrane-cleaving activity. In addition, we revealed the structural dynamics of the transmembrane domains of presenilin during the formation of the complex and its proteolytic process. The SCAM provides new insights into the relationship between the structure and activity of presenilin, and is useful for probing the protein dynamics of the membrane-embedded enzymes.
早老素是γ-分泌酶的催化亚基，它与尼克斯坦、Aph-1和Pen-2等结合辅助因子一起，在脂质双分子层中水解几种跨膜蛋白。然而，这种不寻常的蛋白水解过程的结构基础以及分子机制仍然未知。我们使用半胱氨酸替代可及性方法（SCAM）分析了早老素的结构与功能关系，该方法能够通过巯基试剂对引入到所需位置的半胱氨酸残基的可及性来确定亲水环境。结合小分子抑制剂/调节剂和交联实验，我们能够确定早老素中有助于其膜内切割活性的某些残基和区域。此外，我们揭示了早老素跨膜结构域在复合物形成及其蛋白水解过程中的结构动态。SCAM为早老素的结构与活性之间的关系提供了新的见解，并且有助于探究膜嵌入酶的蛋白质动态。
