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型蛋白酶复合体。这 复合体可切割多种I型完整膜蛋白的跨膜区(TMD) 在它们的胞外区域被脱落酶释放之后。在这些底物中有Notch和淀粉样蛋白β 前体(APP)。TMD裂解Notch受体是后生动物必不可少的细胞信号机制的一部分 生物学和癌症中的失调，以及APP到淀粉样蛋白β-蛋白(Aβ)的TMD切割是必不可少的 阿尔茨海默病(AD)的发病机制。这项提案的首要目标是阐明如何 膜内蛋白分解是由γ-分泌酶完成的。为此，我们将讨论三个主要问题 关于这种在生物学和医学中如此关键的复杂酶的机制，尚无定论。 (1)γ-分泌酶如何识别底物？当酶分解底物的TMD时，我们寻求 了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 酶复合体的结构来设计实验，以确定早老素上的门，从而允许进入 底物TMD进入活性部位。(2)γ-分泌酶如何进行过程性蛋白分解？γ-分泌酶 最初在接近胞质界面的TMD内裂解底物，然后进行 羧基肽酶的修剪会产生分泌肽。我们将讨论较长的Aβ肽是否 与较短的Aβ肽相比，中间体对酶有更高的亲和力，有更多的时间进行修剪。 我们还将解决多肽产品中间体是否与酶结合或解离的问题。 以进一步修剪。(3)γ-分泌酶如何解离蛋白质降解的螺旋TMD底物？我们有 设计了一系列混合螺旋肽/过渡态类似物抑制剂来模拟TMD底物结合 到处于过渡态的酶。与领先的结构生物学实验室合作，结构阐明 结合活性γ-分泌酶的最有效的杂交抑制剂将被确定为了解其结构 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.