Sato Toru, Ananda Kuppanna, Cheng Cathy I, Suh Eric J, Narayanan Saravanakumar, Wolfe Michael S
Center for Neurologic Diseases, Brigham and Women's Hospital and Harvard Medical School, Boston, Massachusetts 02115, USA.
J Biol Chem. 2008 Nov 28;283(48):33287-95. doi: 10.1074/jbc.M805670200. Epub 2008 Oct 1.
Signal peptide peptidase (SPP) and gamma-secretase are intramembrane aspartyl proteases that bear similar active site motifs but with opposite membrane topologies. Both proteases are inhibited by the same aspartyl protease transition-state analogue inhibitors, further evidence that these two enzymes have the same basic cleavage mechanism. Here we report that helical peptide inhibitors designed to mimic SPP substrates and interact with the SPP initial substrate-binding site (the "docking site") inhibit both SPP and gamma-secretase, but with submicromolar potency for SPP. SPP was labeled by helical peptide and transition-state analogue affinity probes but at distinct sites. Nonsteroidal anti-inflammatory drugs, which shift the site of proteolysis by SPP and gamma-secretase, did not affect the labeling of SPP or gamma-secretase by the helical peptide or transition-state analogue probes. On the other hand, another class of previously reported gamma-secretase modulators, naphthyl ketones, inhibited SPP activity as well as selective proteolysis by gamma-secretase. These naphthyl ketones significantly disrupted labeling of SPP by the helical peptide probe but did not block labeling of SPP by the transition-state analogue probe. With respect to gamma-secretase, the naphthyl ketone modulators allowed labeling by the transition-state analogue probe but not the helical peptide probe. Thus, the naphthyl ketones appear to alter the docking sites of both SPP and gamma-secretase. These results indicate that pharmacological effects of the four different classes of inhibitors (transition-state analogues, helical peptides, nonsteroidal anti-inflammatory drugs, and naphthyl ketones) are distinct from each other, and they reveal similarities and differences with how they affect SPP and gamma-secretase.
信号肽肽酶（SPP）和γ-分泌酶是膜内天冬氨酸蛋白酶，它们具有相似的活性位点基序，但膜拓扑结构相反。这两种蛋白酶都被相同的天冬氨酸蛋白酶过渡态类似物抑制剂所抑制，这进一步证明这两种酶具有相同的基本切割机制。在此我们报告，设计用于模拟SPP底物并与SPP初始底物结合位点（“对接位点”）相互作用的螺旋肽抑制剂可同时抑制SPP和γ-分泌酶，但对SPP的抑制效力为亚微摩尔级别。螺旋肽和过渡态类似物亲和探针可标记SPP，但标记位点不同。非甾体抗炎药可改变SPP和γ-分泌酶的蛋白水解位点，但不影响螺旋肽或过渡态类似物探针对SPP或γ-分泌酶的标记。另一方面，另一类先前报道的γ-分泌酶调节剂萘基酮，可抑制SPP活性以及γ-分泌酶的选择性蛋白水解。这些萘基酮显著破坏了螺旋肽探针对SPP的标记，但不阻断过渡态类似物探针对SPP的标记。对于γ-分泌酶，萘基酮调节剂允许过渡态类似物探针对其进行标记，但不允许螺旋肽探针对其进行标记。因此，萘基酮似乎改变了SPP和γ-分泌酶的对接位点。这些结果表明，四类不同抑制剂（过渡态类似物、螺旋肽、非甾体抗炎药和萘基酮）的药理作用彼此不同，并且揭示了它们在影响SPP和γ-分泌酶方面的异同。
