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肽（ABeta）的积累，淀粉样β肽是该疾病特征性脑斑块的主要蛋白质组分。两种酶参与从淀粉样前体蛋白（APP）产生ABeta：BACE，一种细胞外的N-酰基蛋白酶，产生N-末端;称为γ-分泌酶的酶复合物催化膜内蛋白水解步骤，导致形成ABeta 40和ABeta 42的不同C-末端。早老素（PS）蛋白，发现作为在家族性形式的AD的突变位点，含有γ-分泌酶的催化乙酰基残基。虽然尚未开发出针对BACE的有效抑制剂，但有几种有效的γ-分泌酶抑制剂可用。γ-分泌酶的抑制被认为是成功治疗方法的关键;然而，APP不是其唯一的底物。其他几种I型膜蛋白在其假定的跨膜片段内经历γ-分泌酶依赖性蛋白水解，并且其中，蛋白水解对于Notch功能的重要性已得到充分确立。Notch信号在人类健康中起着重要作用。Notch的膜内蛋白水解是其信号传导机制的重要调节组分。在饱和条件下，APP和Notch相互竞争膜内蛋白水解，但不结合PS，这表明多个和可能不同的底物结合位点存在于γ-分泌酶。由于γ-分泌酶的抑制对阿尔茨海默病的治疗有很大的希望，因此我们必须了解γ-分泌酶如何特异性地识别其底物。这将使我们能够设计抑制剂，通过干扰APP/PS相互作用，同时避免干扰其他底物（如Notch）的切割，从而潜在地阻断ABeta的产生。如果能够获得晶体学信息，就有可能预测哪些氨基酸取代会破坏特定的酶-底物界面，同时保留其他重要的酶-底物界面;然而，人们仍然必须对每对这种相互作用进行经验分析-这是一个费力而漫长的过程，不太可能在不久的将来发生。我们提出的结构-活性的方法，将阐明γ-分泌酶内的分子内和分子间的相互作用，γ-分泌酶和其底物之间，从而提供宝贵的信息，有没有良好的高分辨率的结构，这些接口的日期。我们提出的遗传方法是基于功能互补试验，我们还将进行HTS屏幕在一个新的测定直接测量底物-酶的相互作用。

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.