Mapping substrate/gamma-secretase interactions

描述(由申请人提供)： 阿尔茨海默病(AD)发病机制中的一个关键事件是淀粉样β蛋白(ABeta)的积聚，这是该病特有的脑斑块的主要蛋白质成分。从淀粉样前体蛋白(APP)合成ABeta涉及两种酶：BACE，一种胞外天冬氨酸蛋白酶，产生N-末端；称为伽马分泌酶的酶复合体催化膜内蛋白分解步骤，导致Abeta40和Abeta42不同的C-末端的形成。早老素(PS)蛋白是家族性阿尔茨海默病中发现的突变基因，含有γ-分泌酶的天冬氨酸酶催化残基。虽然还没有开发出针对BACE的有效抑制剂，但有几种有效的伽马分泌酶抑制剂可用。抑制γ-分泌酶被认为是成功治疗方法的关键；然而，APP并不是唯一的底物。其他几种I型膜蛋白在其假定的跨膜节段内经历依赖于伽马分泌酶的蛋白分解，其中，蛋白分解对Notch功能的重要性是众所周知的。Notch信号在人类健康中发挥着重要作用。Notch的膜内蛋白降解是其信号转导机制中的重要调控成分。在饱和条件下，APP和Notch相互竞争膜内蛋白降解，但不竞争与PS的结合，这表明伽马分泌酶上存在多个甚至可能是不同的底物结合部位。由于抑制伽玛分泌酶对阿尔茨海默病的治疗有很大的希望，所以我们必须了解伽玛分泌酶是如何识别其底物的。这将使我们能够设计出能够通过干扰APP/PS相互作用而潜在地阻止ABeta产生的抑制剂，同时避免干扰其他底物的切割，如Notch。如果人们能够获得结晶学信息，就有可能预测哪些氨基酸取代会扰乱特定的酶-底物界面，而不会破坏其他重要的酶-底物界面；然而，人们仍然必须对每一对这样的相互作用进行实证分析--这是一个费力而漫长的过程，不太可能在不久的将来发生。我们提出了结构-活性方法，它将揭示伽马分泌酶内部和分子间的相互作用，从而提供宝贵的信息；到目前为止，这些界面还没有良好的高分辨率结构。我们提出的遗传方法是基于功能互补分析的；我们还将在一种新的直接测量底物-酶相互作用的测试中进行HTS筛选。

DESCRIPTION (provided by applicant): A key event in the pathogenesis of Alzheimer's disease (AD) is the accumulation of amyloid-Beta peptide (ABeta), the principal protein component of the cerebral plaques characteristic of this disease. Two enzymes are involved in generating ABeta from the Amyloid Precursor Protein (APP): BACE, an extracellular aspartyl protease, generates the N-terminus; the enzymatic complex called gamma-secretase catalyzes the intramembrane proteolysis step resulting in the formation of the divergent C-termini of ABeta40 and ABeta42. Presenilin (PS) proteins, discovered as mutated loci in familial forms of AD, contain the catalytic aspartyl residues of gamma-secretase. While no potent inhibitors have been developed against BACE, several potent gamma-secretase inhibitors are available. Inhibition of gamma-secretase is thought to be key to successful therapeutic approaches; however, APP is not its only substrate. Several other Type I membrane proteins undergo gamma-secretase-dependent proteolysis within their putative membrane spanning segments and of these, the importance of proteolysis for Notch function is well established. Notch signaling plays an important role in human health. Intramembrane proteolysis of Notch is an essential regulated component of its signaling mechanism. Under saturating conditions, APP and Notch compete with each other for intramembrane proteolysis, but not for binding to PS, suggesting that multiple and perhaps distinct substrate binding sites exist on gamma-secretase. Since inhibition of gamma-secretase holds great promise for the treatment of Alzheimer's disease, it is essential we understand how gamma-secretase specifically recognizes its substrates. This will enable us to design inhibitors that can potentially block ABeta production by interfering with APP/PS interactions while avoiding interference with the cleavage of other substrates such as Notch. If one had access to crystallographic information, it would be possible to predict which amino acid substitutions would disrupt specific enzyme-substrate interfaces while sparing other important enzyme-substrate interfaces; however, one would still have to carry out empirical analyses for each pair of such interactions - a laborious and lengthy process that is unlikely to happen in the immediate future. We propose structure-activity approach that will shed light on the intramolecular and intermolecular interactions within gamma-secretase and between gamma-secretase and its substrates thus providing invaluable information; there is no good high-resolution structure of these interfaces to date. The genetic approach we propose is based on functional complementation assays; we will also conduct HTS screens in a novel assay directly measuring substrate-enzyme interactions.