Deutsches Zentrum für Neurodegenerative Erkrankungen (DZNE) and Adolf-Butenandt-Institute, Biochemistry, Ludwig-Maximilians-University, München, Germany.
J Neurochem. 2010 Feb;112(4):940-50. doi: 10.1111/j.1471-4159.2009.06510.x. Epub 2009 Dec 15.
gamma-Secretase is a pivotal intramembrane-cleaving protease complex and important drug target for Alzheimer's disease. The protease not only releases small peptides, such as the amyloid-beta peptide, which drives Alzheimer's disease pathogenesis, but also intracellular domains, which can have critical functions in nuclear signaling. Unlike typical aspartyl proteases, gamma-secretase contains a non-classical GxGD active site motif in its catalytic subunit presenilin (PS) 1 or PS2. It is not known whether both glycines are of similar functional relevance and why the glycine residues are invariant elements of the motif. Here we identify the N-terminal glycine of the GxGD motif in PS1, G382, as a critical residue of the active site domain of gamma-secretase. Substitution of G382 by a number of different amino acids abrogated gamma-secretase activity. Only the smallest possible G382A substitution allowed substantial gamma-secretase activity. Depending on the substrate, however, the presence of G382 could become even an absolute functional requirement of gamma-secretase. Very similar results were obtained for the C-terminal glycine residue (G384) of the GxGD motif. Our data thus identify a requirement for small side chain residues in the active site domain of gamma-secretase and suggest that the glycines of the GxGD motif could be evolutionary conserved to allow cleavage of all possible gamma-secretase substrates, including those, which are highly sensitive to minimal alteration of the PS active site domain. These findings broaden our understanding of gamma-secretase substrate recognition and cleavage, which may prove crucial for therapeutic targeting of the enzyme.
γ-分泌酶是一种重要的跨膜切割蛋白酶复合物，也是阿尔茨海默病的重要药物靶点。该蛋白酶不仅释放小肽，如驱动阿尔茨海默病发病机制的淀粉样β肽，还释放细胞内结构域，这些结构域在核信号中具有关键功能。与典型的天冬氨酸蛋白酶不同，γ-分泌酶在其催化亚基早老素（PS）1 或 PS2 中含有非经典的 GxGD 活性位点基序。目前尚不清楚两个甘氨酸是否具有相似的功能相关性，以及为什么甘氨酸残基是该基序不变的组成部分。在这里，我们确定 PS1 中 GxGD 基序的 N 端甘氨酸 G382 是 γ-分泌酶活性位点域的关键残基。用许多不同的氨基酸替代 G382 会使 γ-分泌酶活性丧失。只有 G382A 最小的取代才能允许大量的 γ-分泌酶活性。然而，取决于底物，G382 的存在甚至可能成为 γ-分泌酶的绝对功能要求。对于 GxGD 基序的 C 端甘氨酸残基（G384）也得到了非常相似的结果。因此，我们的数据确定了 γ-分泌酶活性位点域中小侧链残基的需求，并表明 GxGD 基序中的甘氨酸残基可能是进化保守的，以允许切割所有可能的 γ-分泌酶底物，包括那些对 PS 活性位点域的最小改变高度敏感的底物。这些发现拓宽了我们对 γ-分泌酶底物识别和切割的理解，这对于酶的治疗靶向可能至关重要。
