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: 2016
• 资助国家: 加拿大
• 起止时间: 2016-01-01 至 2017-12-31
• 项目状态: 已结题
• 来源: https://www.nserc-crsng.gc.ca/ase-oro/Details-Detailles_eng.asp?id=613156
• 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.

这项发现资助的重点是我们对脱落酶的独立、长期研究，脱落酶是一类重要的酶，可促进生长和发育中的“胞外域脱落”。目前，我们的模型脱落酶是 BACE1，一种与胃蛋白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。单个跨膜序列 (TMS) 将胞外域与胞质尾连接，构成天冬氨酸蛋白酶的独特特征。最近的文献表明，BACE1 可以切割比预期更多样化的底物。由于我们最近的研究结果还表明金属离子可以与 BACE1 相互作用，因此我们假设铜调节其生理特性。 目标 该研究计划使用各种生化和生物物理工具，研究脱落酶超出其典型酶促功能的多种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，以及金属离子如何影响其体外和体内功能。具体来说，我们将： 1) 表达重组 BACE1 以鉴定和表征其金属离子结合位点； 2) 确定两个 BACE1 构建体（即 #1，胞外域；#2，TMS 和胞质域）在铜不存在和存在的情况下的 3D 结构； 3) 评估铜作为 BACE1 配体的体内重要性。 方法论 细胞 (HEK293T) 和无细胞系统都将用于表达我们的模型蛋白 BACE1。亲和纯化的 BACE1 将进行有限的蛋白水解和质谱分析，以鉴定金属离子结合残基和诱导的构象效应。亚基计数和 FLIM-FRET 将用于评估活细胞中的 BACE1 同源相互作用。纯化的 BACE1 片段将进行体外结构分析（NMR 和 X 射线晶体学），BACE1-铜复合物将进行体内检查（果蝇和 Long-Evans Cinnamon 大鼠）。 新颖性和影响力 该 sheddase 计划包含三个创新技术方面。使用 BACE1 作为我们当前的模型，亚基计数将确定其在活细胞中的化学计量。其次，一种新型的无细胞蛋白质合成系统将允许对 BACE1 TMS 进行分析。第三，一种新的果蝇模型将允许在体内研究 BACE1-铜复合物。鉴于脱落酶的结构和多方面功能在很大程度上尚未被探索，该研究计划（以及前沿资助提案）中最先进的目标和方法为表征膜中蛋白质相互作用提供了一个新的范例。我们对脱落酶的详细分析将 NSERC 及其 HQP 学员在胞外域脱落生物学这一基本领域中的独特定位，将帮助未来的工业合作伙伴操纵其不同的生理作用。