STRUCTURE, FUNCTION AND REGULATION OF GAMMA-SECRETASE

早老素(PS)作为第一种膜内天冬氨酸蛋白酶和γ-分泌酶催化中心的发现 发生在项目1中。从那时起，已经确定了&gt；100种底物。PS/γ-分泌酶介导 所有后生动物生命所必需的关键信号通路及其APP的裂解释放淀粉样蛋白 在所有AD患者体内积聚的B蛋白。在确定PS是一种蛋白酶后，项目1已经 继续为PS生物学做出积极贡献。我们提出--并提供了第一个证据--全息图 经过自催化内蛋白分解生成活性杂二聚体，第一次重组的PS及其3 哺乳动物细胞中的辅因子，将该酶纯化为均一，获得了yComplex的第一个三维结构。 通过EM，进行了SILAC筛选，确定了几种新的底物，并设计了许多伽马分泌酶 抑制剂，其中一些是有效的，比在 人类。该项目现在进行了修订，以回应SEP的所有有益批评。我们建议 研究伽玛分泌酶生物化学中的4个相关课题。1：一种新的分泌酶细胞生物学模型 处理我们发现，与目前的概念相反，α-(亚当10)、β-和伽马-分泌酶存在 部分是在一个大的蛋白质复合体中，它可以调节底物的有效顺序处理。我们的 广泛的支持数据包括来自wt脑的内源性α-和伽马-分泌酶的稳健的共IP以及 APP基板的顺序阿尔法/伽马处理。我们建议充分确认这一新的监管模式 膜内蛋白分解，并询问它是否推广到另一膜蛋白水解酶对：S1P/S2P。2：The 膜脂对γ-分泌酶的复杂调控。我们将扩大我们最近的证据，证明某些人头 膜脂的基团和脂肪酰基侧链有效地上调和下调伽马裂解，包括 关键字A(I42/40比率。我们将试图通过操纵某些脂质的体外效应来验证其强大的体外效应。 体内同源生物合成和分解代谢酶。3：迈向更高的伽马分泌酶的结构分辨率 Complex与领先的结构生物学家合作，我们将追求具有技术挑战性的 对伽马分泌酶结构的基本探索：a)2D晶体的进一步冷冻-EM分析；b)3D x射线 单个伽马成分(PS、Net、Pen-2)的结晶学；以及c)尝试进行3DX射线结晶学 纯化的全息酶。4：提炼有效和选择性的避免凹槽的伽马抑制剂，并确定其 机制。在我们已经合成的1600多种化合物的基础上，我们将开发出抑制SARS的药物 App与Notch，对于最具选择性的化合物，评估其他伽马底物的裂解并测试它们 在老鼠身上。这些目标建立在我们的经验基础上，以解决伽马分泌生物学中一些最棘手的问题。

The discovery of presenilin (PS) as the first intramembrane aspartyl protease and the catalytic center of gamma-secretase occurred in Project 1. Since then, >100 substrates have been identified. PS/gamma-secretase mediates critical signaling pathways necessary for life in all metazoans, and its cleavage of APP releases the amyloid B-protein that accumulates in all patients with AD. After identifying PS as a protease, Project 1 has continued to contribute actively to PS biology. We proposed - and provided the first evidence - that holoPS undergoes autocatalytic endoproteolysis to generate the active heterodimer, first reconstituted PS and its 3 cofactors in mammalian cells, purified the protease to homogeneity, obtained the first 3D structure of the ycomplex by EM, conducted SILAC screens to identify several new substrates, and designed many gamma-secretase inhibitors, some of which are potent and much more APP-selective than compounds tried in humans. The Project is now revised to respond to all of the SEP's helpful critiques. We propose to study 4 related topics in the biochemistry of gamma-secretase. 1: A new cell biological model of secretase processing We have discovered that contrary to current concepts, alpha- (ADAM 10), beta- and gamma-secretases exist in part in a large protein complex that can mediate efficient sequential processing of substrates. Our extensive supporting data include robust co-IP of endogenous a- and gamma-secretases from wt brain and the sequential alpha/gamma processing of an APP substrate. We propose to fully confirm this new model of regulated intramembrane proteolysis and ask if it generalizes to another membrane protease pair: S1P/S2P. 2: The complex regulation of gamma-secretase by membrane lipids. We will extend our recent evidence that certain head groups and fatty acyl side chains of membrane lipids potently up- and down-regulate gamma-cleavages, including the key A(i42/40 ratio. We'll seek to validate robust in vitro effects of certain lipids by manipulating their cognate biosynthetic and catabolic enzymes in vivo. 3: Toward greater structural resolution of the gamma-secretase complex Working with leading structural biologists, we will pursue the technically challenging but essential quest for the structure of gamma-secretase via: a) further cryo-EM analyses of 2D crystals; b) 3D x-ray crystallography of individual gamma-components (PS, Net, Pen-2); and c) attempted 3D x-ray crystallography of the purified holo-enzyme. 4: Refining potent and selective Notch-sparing gamma-inhibitors and defining their mechanism. Building on more than 1,600 compounds we've synthesized, we will develop SARs for inhibiting APP vs. Notch, and for the most selective compounds, assess cleavage of other gamma-substrates and test them in mice. These aims build on our experience to tackle some of the thorniest problems in gamma-secretase biology.