Mechanisms of Gamma-Secretase

项目摘要 γ-分泌酶是一种以早老素为催化组分的膜包埋蛋白酶复合物。这 复合物切割多种I型整合膜蛋白的跨膜结构域（TMD 在它们的胞外结构域被脱落酶释放后。这些底物包括Notch和淀粉样β蛋白 前体（APP）。Notch受体的TMD切割是后生动物必需的细胞信号传导机制的一部分 在癌症中，APP的TMD裂解为淀粉样β蛋白（Aβ）是生物学和失调的关键， 阿尔茨海默病（AD）的发病机制。本提案的总体目标是阐明如何 膜内蛋白水解由γ-分泌酶完成。为此，我们将在三个中心 关于这种在生物学和医学中至关重要的复杂酶的机制的公开问题。 (1)γ-分泌酶如何识别底物？当酶切割底物的TMD时，我们试图 理解螺旋度在底物识别中的作用。此外，我们还使用了最近的高分辨率 结构的蛋白酶复合物，以设计实验，以确定门早老蛋白，允许访问 底物TMD进入活性位点。(2)γ-分泌酶是如何进行蛋白质降解的？γ-分泌酶 最初在靠近胞质界面的TMD内切割底物，随后进行性切割， 羧肽酶修剪导致分泌肽。我们将讨论是否较长的Aβ肽 与较短的Aβ肽相比，中间体对酶具有更高的亲和力，允许更多的时间进行修剪。 我们还将讨论肽产物中间体在酶作用之前是否保持结合或解离。 进一步修剪。(3)γ-分泌酶如何解螺旋TMD底物以进行蛋白质水解？我们有 设计了一系列混合螺旋肽/过渡态类似物抑制剂，以模拟TMD底物结合 过渡态的酶。与领先的结构生物学实验室合作， 将确定与活性γ-分泌酶结合的最有效的杂合抑制剂的结构， γ-分泌酶作用机制的基础。

Project Summary γ-Secretase is a membrane-embedded protease complex with presenilin as its catalytic component. This complex cleaves the transmembrane domains (TMDs) of a wide variety of type I integral membrane proteins after their ectodomain release by sheddases. Among these substrates are Notch and the amyloid β-protein precursor (APP). TMD cleavage of Notch receptors is part of cell signaling mechanisms essential to metazoan biology and dysregulated in cancer, and TMD cleavage of APP to amyloid β-protein (Aβ) is essential to the pathogenesis of Alzheimer's disease (AD). The overarching goal of this proposal is to elucidate how intramembrane proteolysis is accomplished by γ-secretase. Toward this end, we will address three central open questions regarding the mechanism of this complex enzyme that is so critical in biology and medicine. (1) How does γ-secretase recognize substrates? As the enzyme cleaves the TMD of substrate, we seek to understand the role of helicity in substrate recognition. In addition, we have used a recent high-resolution structure of the protease complex to design experiments to identify the gate on presenilin that allows access of substrate TMD into the active site. (2) How does γ-secretase carry out processive proteolysis? γ-Secretase initially cleaves substrates within their TMDs close to the cytosolic interface, followed by processive carboxypeptidase trimming resulting in secreted peptides. We will address whether the longer Aβ peptide intermediates have higher affinity for the enzyme, allowing more time for trimming, than shorter Aβ peptides. We will also address whether peptide product intermediates remain bound or dissociate from the enzyme prior to further trimming. (3) How does γ-secretase unwind helical TMD substrates for proteolysis? We have designed a series of hybrid helical peptide/transition-state analog inhibitors to mimic the TMD substrate bound to the enzyme in the transition state. In collaboration with a leading structural biology lab, structure elucidation of the most potent hybrid inhibitor bound to active γ-secretase will be determined to understand the structural basis of the γ-secretase mechanism of action.