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.

项目摘要 γ-分泌酶是一种膜上包含的蛋白酶复合物，其催化剂是其催化成分。这复合物裂解多种I型积分膜蛋白的跨膜结构域（TMD）在其外部域通过Sheddase释放后。在这些底物中是Notch和淀粉样蛋白β-蛋白前体（APP）。 Notch受体的TMD切割是后生动物必不可少的细胞信号传导机制的一部分在癌症中生物学和失调，APP对淀粉样蛋白β-蛋白（Aβ）的TMD切割对于阿尔茨海默氏病的发病机理（AD）。该提案的总体目标是阐明如何膜内蛋白水解是通过γ-分泌酶完成的。为此，我们将解决三个中央关于这种复杂酶在生物学和医学中至关重要的复杂酶的机制的开放问题。（1）γ-分泌酶如何识别底物？随着酶裂解底物的TMD，我们寻求了解螺旋在底物识别中的作用。此外，我们使用了最近的高分辨率蛋白酶复合物的结构以设计实验，以识别允许访问基板TMD进入活动站点。（2）γ-分泌酶如何进行过程蛋白水解？ γ-分泌酶最初将其在其TMD靠近胞质界面的TMD中切割，然后是过程羧肽酶修剪导致分泌的肽。我们将解决较长的Aβ辣椒中间体对酶具有更高的亲和力，比较短的Aβ肽允许修剪时间更多。我们还将解决肽产品中间体是否保持绑定或与酶解离（3）γ-分泌酶如何解开蛋白水解的螺旋TMD底物？我们有设计了一系列杂化螺旋肽/过渡态类似物抑制剂，以模仿TMD底物结合到过渡状态的酶。与领先的结构生物学实验室合作，结构阐明将确定与活性γ-分泌酶结合的最有效的混合抑制剂中作用机理的基础。