Department of Developmental Biology, Washington University School of Medicine, St. Louis, Missouri 63110, USA.
J Neurosci. 2010 Feb 3;30(5):1648-56. doi: 10.1523/JNEUROSCI.3826-09.2010.
Gamma-secretase is a multiprotein, intramembrane-cleaving protease with a growing list of protein substrates, including the Notch receptors and the amyloid precursor protein. The four components of gamma-secretase complex--presenilin (PS), nicastrin (NCT), Pen2, and Aph1--are all thought to be essential for activity. The catalytic domain resides within PS proteins, NCT has been suggested to be critical for substrate recognition, and the contributions of Pen2 and Aph1 remain unclear. The role of NCT has been challenged recently by the observation that a critical residue (E332) in NCT, which had been thought to be essential for gamma-secretase activity, is instead involved in complex maturation. Here, we report that NCT is dispensable for gamma-secretase activity. NCT-independent gamma-secretase activity can be detected in two independent NCT-deficient mouse embryonic fibroblast lines and blocked by the gamma-secretase inhibitors N-[N-(3,5-difluorophenacetyl-L-alanyl)]-S-phenylglycine t-butyl ester and L-685,458. This catalytic activity requires prior ectodomain shedding of the substrate and can cleave ligand-activated endogenous Notch receptors, indicating presence of this activity at the plasma membrane. Small interfering RNA knockdown experiments demonstrated that NCT-independent gamma-secretase activity requires the presence of PS1, Pen2, and Aph1a but can tolerate knockdown of PS2 or Aph1b. We conclude that a PS1/Pen2/Aph1a trimeric complex is an active enzyme, displaying biochemical properties similar to those of gamma-secretase and roughly 50% of its activity when normalized to PS1 N-terminal fragment levels. This PS1/Pen2/Aph1a complex, however, is highly unstable. Thus, NCT acts to stabilize gamma-secretase but is not required for substrate recognition.
γ-分泌酶是一种多蛋白跨膜切割蛋白酶，其具有越来越多的蛋白底物，包括 Notch 受体和淀粉样前体蛋白。γ-分泌酶复合物的四个组成部分——早老素（PS）、尼卡斯特林（NCT）、Pen2 和 Aph1——都被认为是活性所必需的。催化结构域位于 PS 蛋白内，NCT 被认为对于底物识别至关重要，而 Pen2 和 Aph1 的贡献仍不清楚。最近的观察结果对 NCT 的作用提出了挑战，即 NCT 中的一个关键残基（E332）曾被认为对于 γ-分泌酶活性是必需的，但实际上它参与了复合物的成熟。在这里，我们报告 NCT 对于 γ-分泌酶活性不是必需的。在两个独立的 NCT 缺陷型小鼠胚胎成纤维细胞系中可以检测到 NCT 非依赖性 γ-分泌酶活性，并被 γ-分泌酶抑制剂 N-[N-(3,5-二氟苯乙酰基)-L-苯丙氨酸]-S-苯基甘氨酸叔丁酯和 L-685,458 阻断。这种催化活性需要底物的前外显子脱落，并能切割配体激活的内源性 Notch 受体，表明这种活性存在于质膜上。小干扰 RNA 敲低实验表明，NCT 非依赖性 γ-分泌酶活性需要 PS1、Pen2 和 Aph1a 的存在，但可以耐受 PS2 或 Aph1b 的敲低。我们得出结论，PS1/Pen2/Aph1a 三聚体复合物是一种活性酶，具有与 γ-分泌酶相似的生化特性，当与 PS1 N 端片段水平归一化时，其活性约为 50%。然而，这种 PS1/Pen2/Aph1a 复合物非常不稳定。因此，NCT 作用于稳定 γ-分泌酶，但对于底物识别不是必需的。
