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Mechanisms of Gamma-Secretase

γ-分泌酶的机制

基本信息

批准号：
10004095
负责人：
Michael S Wolfe
金额：
$ 29.16万
依托单位：
UNIVERSITY OF KANSAS LAWRENCE
依托单位国家：
美国
项目类别：
财政年份：
2017
资助国家：
美国
起止时间：
2017-09-20 至 2022-08-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10004095
关键词：
Active Sites Address Affinity Alanine Alzheimer&apos s Disease Amino Acids Amyloid beta-Protein Amyloid beta-Protein Precursor Architecture Aspartate Binding Biochemical Biology C-terminal Carboxypeptidase Chemicals Cleaved cell Collaborations Complex Cryoelectron Microscopy Crystallization Cysteine Disease Dissociation Disulfides Docking Enzymes Goals Homologous Gene Hybrids Integral Membrane Protein Laboratories Length Lipid Bilayers Malignant Neoplasms Medicine Membrane Membrane Proteins Molecular Conformation Multienzyme Complexes Mutagenesis Pathogenesis Peptide Hydrolases Peptides Play Proteins Proteolysis Resolution Role Series Signal Transduction Site Structure Structure-Activity Relationship Substrate Domain Surface Plasmon Resonance Techniques Testing Time Transmembrane Domain Universities Water Work analog design experimental study gamma secretase inhibitor/antagonist notch protein presenilin rhomboid structural biology

项目摘要

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.

项目概要 γ-Secretase 是一种膜嵌入的蛋白酶复合物，以早老素为催化成分。这复合物可切割多种 I 型整合膜蛋白的跨膜结构域 (TMD) 在它们的胞外域被脱落酶释放后。这些底物包括 Notch 和 β 淀粉样蛋白前体（APP）。 Notch 受体的 TMD 裂解是后生动物必需的细胞信号传导机制的一部分 APP 与淀粉样 β 蛋白 (Aβ) 的 TMD 切割对于癌症的生物学和失调至关重要阿尔茨海默病（AD）的发病机制。该提案的总体目标是阐明如何膜内蛋白水解是由γ-分泌酶完成的。为此，我们将解决三个核心问题：关于这种在生物学和医学中至关重要的复杂酶的机制尚有待解决的问题。 (1) γ-分泌酶如何识别底物？当酶裂解底物的 TMD 时，我们寻求了解螺旋度在底物识别中的作用。此外，我们还使用了最新的高分辨率蛋白酶复合物的结构，以设计实验来识别早老素上允许访问的门底物TMD进入活性位点。 (2) γ-分泌酶如何进行持续蛋白水解？ γ-分泌酶最初在靠近胞质界面的 TMD 内裂解底物，然后进行持续羧肽酶修剪导致分泌肽。我们将解决是否较长的 Aβ 肽与较短的 Aβ 肽相比，中间体对酶具有更高的亲和力，因此可以有更多的时间进行修剪。我们还将解决肽产品中间体在酶之前是否保持结合或解离的问题。以便进一步修剪。 (3) γ-分泌酶如何解旋螺旋TMD底物进行蛋白水解？我们有设计了一系列混合螺旋肽/过渡态类似物抑制剂来模拟 TMD 底物结合到过渡态的酶。与领先的结构生物学实验室合作，进行结构解析将确定与活性γ-分泌酶结合的最有效的杂合抑制剂的结构，以了解其结构 γ-分泌酶作用机制的基础。