Investigating the role of Reep5 in formation, maintenance, and function of the endoplasmic reticulum

研究 Reep5 在内质网形成、维持和功能中的作用

• 批准号: RGPIN-2016-05618
• 负责人: Gramolini, Anthony
• 金额: $ 2.77万
• 依托单位: University of Toronto
• 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=615807
• 关键词: Investigating; role; Reep5; formation; maintenance

The Endoplasmic Reticulum (ER) is a dynamic multi-functional organelle found in eukaryotes. It is a specialized organ responsible for many functions, including Ca2+ cycling, protein translation and trafficking. An important group of proteins that mediate formation and maintenance of the ER are proteins with ‘reticulon homology domains (RHD). One group of RHD proteins are the receptor expression-enhancing proteins (REEPs). The precise role of REEPs in ER formation, maintenance, and responses to ER stress is not well understood. Our ongoing proteomic studies have identified REEP5 to be an evolutionary-conserved, cardiac cell-enriched, membrane protein that is the dominant REEP in the heart. Hypothesis and Aim: We hypothesize that REEP5 controls the ER homeostatic environment and ER integrity, affecting cardiac function through its regulation of Ca2+ signaling, and is centrally involved in cardiac ER stress responses. My primary goal is to investigate the mechanism of how REEP5 contributes to normal ER formation, maintenance and function. We propose utilizing cell biological, biochemical, mass spectrometry, and advanced cellular imaging approaches to address the role of REEP5. Examine the effect of Reep5 knockdown on HEK and myocyte ER. We will perform immunofluorescence and super resolution microscopy to examine REEP5-localization in cells. Site-directed mutation of REEP5 will be performed and consequences of mutations will be assessed on ER morphology and localization. To study ER stress responses, we will assess WT and knockdown of REEP5 using lentiviral vectors with/without ER stress induction induced by H2O2 and tunicamycin. Reactive oxygen species (ROS) reflecting ER stress, will be measured, as will cell viability and apoptosis. Identify REEP5 interacting proteins. Using standard immunoprecipitation studies and mass spectrometry we will identify REEP5 interacting proteins involved in ER formation and maintenance. We generated a REEP5-myc-flag-his expressing stable HEK cell line, and will purify REEP5 and interactors. Analyses will be performed in the presence and absence of ER stresses. Immobilized proteins will be visualized by Coomassie staining and identified by mass spectrometry. Examine the effect of REEP5 deletion in the zebrafish. To investigate the role of REEP5 in heart development, we have generated a morpholino zebrafish for REEP5. REEP5 is expressed in the zebrafish myocardium during embryonic development, and the zebrafish REEP5 protein has high homology (~80%) to the human REEP5. We have successfully silenced REEP5 expression in zebrafish. The initial characterization of the morphants show perturbed embryonic development with abnormal heart formation and dysfunction. Fish and tissues will be assessed by immunoblotting, immunofluorescence, histology and bright field imaging.

内质网（Endoplasmic Reticulum，ER）是真核生物中一种动态的多功能细胞器。它是一个专门的器官，负责许多功能，包括Ca 2+循环，蛋白质翻译和运输。一组重要的介导ER形成和维持的蛋白质是具有网状蛋白同源结构域（RHD）的蛋白质。一组RHD蛋白是受体表达增强蛋白（REEP）。REEPs在ER形成、维持和对ER应激的反应中的确切作用尚不清楚。我们正在进行的蛋白质组学研究已经确定REEP 5是一种进化保守的、富含心脏细胞的膜蛋白，是心脏中占主导地位的REEP。 假设和目的：我们假设REEP 5控制ER稳态环境和ER完整性，通过其对Ca 2+信号的调节影响心脏功能，并主要参与心脏ER应激反应。我的主要目标是研究REEP 5如何有助于正常ER形成，维持和功能的机制。我们建议利用细胞生物学，生物化学，质谱和先进的细胞成像方法来解决REEP 5的作用。 检查Reep 5敲低对HEK和肌细胞ER的影响。我们将进行免疫荧光和超分辨率显微镜检查REEP 5在细胞中的定位。将进行REEP 5的定点突变，并评估突变对ER形态和定位的影响。为了研究ER应激反应，我们将使用慢病毒载体评估WT和REEP 5的敲低，所述慢病毒载体具有/不具有由H2 O2和衣霉素诱导的ER应激诱导。将测量反映ER应激的活性氧（ROS），以及细胞活力和凋亡。 识别REEP 5相互作用蛋白。使用标准 免疫沉淀研究和质谱分析，我们将确定REEP 5 参与ER形成和维持的相互作用蛋白质。我们 产生表达REEP 5-myc-flag-his的稳定HEK细胞系，并且将 纯化REEP 5和相互作用子。分析将在 存在和不存在ER应力。固定化蛋白质将被 通过考马斯染色可视化并通过质谱法鉴定。 研究REEP 5缺失对斑马鱼的影响。为了研究REEP 5在心脏发育中的作用，我们已经产生了一个吗啉代斑马鱼的REEP 5。REEP 5在胚胎发育期间在斑马鱼心肌中表达，并且斑马鱼REEP 5蛋白与人REEP 5具有高度同源性（~80%）。我们已经成功地沉默了斑马鱼中的REEP 5表达。最初的特点的morphants显示干扰胚胎发育异常的心脏形成和功能障碍。鱼和组织将通过免疫印迹、免疫荧光、组织学和明场成像进行评估。