Molelcular Dissection of Ectodomain Shedding: Biology of BACE1 and Other Sheddases.

胞外域脱落的分子解剖：BACE1 和其他脱落酶的生物学。

• 批准号: RGPIN-2015-04774
• 负责人: Multhaup, Gerhard
• 金额: $ 2.77万
• 依托单位: McGill University
• 依托单位国家: 加拿大
• 项目类别: Discovery Grants Program - Individual
• 财政年份: 2019
• 资助国家: 加拿大
• 起止时间: 2019-01-01 至 2020-12-31
• 项目状态: 已结题
• 来源: https://www.nserc-crsng.gc.ca/ase-oro/Details-Detailles_eng.asp?id=687168
• 关键词: Molelcular; Dissection; Ectodomain; Shedding; Biology

This Discovery Grant focuses upon our independent, long-term study of sheddases, an important class of enzymes that facilitate "ectodomain shedding" in growth and development. Currently, our model sheddase is BACE1, a type I transmembrane protein related to the pepsin and retroviral aspartic acid protease families. A single transmembrane sequence (TMS) links the ectodomain to the cytosolic tail, constituting a unique feature amongst aspartic proteases. Recent literature has revealed that BACE1 can cleave a more diverse array of substrates than anticipated. Since our recent findings have also revealed that metal ions can interact with BACE1, we hypothesize that copper modulates its physiological properties.***Objectives***Using a variety of biochemical and biophysical tools, this research program examines the diverse physiological roles and molecular mechanisms of sheddases beyond their canonical enzymatic function. For our model system, the goals include characterizing the tertiary and quaternary structure of BACE1, as well as how metal ions affect its function in vitro and in vivo. Specifically, we will: 1) express recombinant BACE1 to identify and characterize its metal ion-binding site(s); 2) determine the 3D structure of two BACE1 constructs (i.e. #1, ectodomain; #2, TMS and cytoplasmic domain) in the absence and presence of copper; and 3) assess the in vivo importance of copper as a BACE1 ligand.****Methodology****Both cellular (HEK293T) and cell-free systems will be used to express our model protein BACE1. Affinity-purified BACE1 will be subjected to limited proteolysis and Mass Spectrometry to identify metal ion-binding residues and induced conformational effects. Subunit counting and FLIM-FRET will be used to assess BACE1 homo-interactions in living cells. Purified BACE1 fragments will be subjected to in vitro structural analyses (NMR and X-ray Crystallography), and BACE1-copper complexes will be examined in vivo (Drosophila, and Long-Evans Cinnamon rats).****Novelty & Impact***There are three innovative technological aspects within this sheddase program. Using BACE1 as our current model, subunit counting will determine its stoichiometry in living cells. Secondly, a novel cell-free protein synthesis system will allow for analyses of the BACE1 TMS. Thirdly, a novel Drosophila model will allow for the study of BACE1-copper complexes in vivo. Given that the structure and multifaceted functions of sheddases remain largely unexplored, the state-of-the-art objectives and methodologies within this research program (and leading-edge grant proposal) provide a new paradigm for characterizing protein interactions in membranes. Uniquely positioning NSERC and its HQP trainees in this fundamental area of ectodomain shedding biology, our detailed analyses of sheddases will help future industrial partners to manipulate their diverse physiological roles.***************

这笔发现基金专注于我们对脱落酶的独立、长期的研究，脱落酶是一类重要的酶，有助于生长和发育中的“胞外脱落酶”。目前，我们的模型脱落酶是BACE1，一种与胃酶和逆转录病毒天冬氨酸蛋白酶家族相关的I型跨膜蛋白。一个单一的跨膜序列(TMS)将胞外结构域连接到胞浆尾部，构成了天冬氨酸蛋白酶的一个独特特征。最近的文献表明，BACE1可以切割比预期更多样化的底物阵列。由于我们最近的发现也揭示了金属离子可以与BACE1相互作用，我们假设铜调节其生理特性。*目的*本研究计划利用各种生化和生物物理工具，除了其典型的酶功能外，还研究脱落酶的不同生理作用和分子机制。对于我们的模型系统，目标包括表征BACE1的三级和四级结构，以及金属离子如何在体外和体内影响其功能。具体地说，我们将：1)表达重组BACE1以鉴定和鉴定其金属离子结合部位(S)；2)在无铜和有铜存在的情况下确定两个BACE1结构域(即#1，胞外结构域；#2，TMS和细胞质结构域)的三维结构；以及3)评估铜作为BACE1配体在体内的重要性。*方法学*将使用细胞(HEK293T)和非细胞系统来表达我们的模型蛋白BACE1。亲和纯化的BACE1将进行有限的蛋白质降解和质谱分析，以确定金属离子结合残基和诱导构象效应。亚基计数和Flim-FRET将用于评估活细胞中BACE1的同源相互作用。纯化的BACE1片段将进行体外结构分析(核磁共振和X射线结晶学)，BACE1-铜配合物将在体内(果蝇和Long-Evans Cinnamon大鼠)进行检测。*新颖性和影响*这个脱落酶计划有三个创新的技术方面。使用BACE1作为我们目前的生物模型，亚单位计数将确定其在活细胞中的化学计量比。其次，一种新的无细胞蛋白质合成系统将支持对BACE1 TMS的分析。第三，一种新的果蝇模型将允许在体内研究BACE1-铜络合物。鉴于脱落酶的结构和多方面功能在很大程度上仍未被探索，该研究计划中最先进的目标和方法(以及前沿拨款提案)为表征膜中蛋白质的相互作用提供了一个新的范例。NSERC及其HQP学员在这个外域脱落生物学的基本领域具有独特的定位，我们对脱落酶的详细分析将帮助未来的工业合作伙伴操纵他们不同的生理角色。