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

内质网(ER)是真核生物中发现的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，包括钙循环、蛋白质翻译和运输。调节内质网形成和维持的一组重要蛋白质是具有网状同源结构域(RHD)的蛋白质。其中一组RHD蛋白是受体表达增强蛋白(REEP)。Reep在内质网形成、维持和对内质网应激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证实，REEP5是一种进化保守的、富含心肌细胞的膜蛋白，是心脏中主要的REEP。*假设和目的：我们假设REEP5控制内质网稳态环境和内质网完整性，通过其对钙信号的调节来影响心功能，并集中参与心脏内质网应激反应。我的主要目标是研究REEP5如何促进正常内质网的形成、维持和功能。我们建议利用细胞生物学、生化、质谱学和先进的细胞成像方法来研究REEP5的作用。*检测Reep5基因敲除对HEK和心肌细胞ER的影响。我们将进行免疫荧光和超分辨显微镜检查REEP5在细胞中的定位。将进行REEP5的定点突变，并将评估突变对内质网形态和定位的影响。为了研究内质网应激反应，我们将使用慢病毒载体评估在过氧化氢和衣霉素诱导和不诱导内质网应激的情况下REEP5的WT和敲除。将测量反映内质网应激的活性氧簇(ROS)，以及细胞存活率和凋亡率。*识别REEP5相互作用蛋白。利用标准免疫沉淀研究和质谱学，我们将鉴定参与内质网形成和维持的REEP5*相互作用蛋白。我们建立了一个稳定表达REEP5-myc-FLAG-HIS的HEK细胞系，并将纯化REEP5及其相互作用因子。分析将在存在和不存在ER应力的情况下进行。固定化的蛋白质将通过考马斯亮染和质谱学鉴定。*检测REEP5在斑马鱼中缺失的影响。为了研究REEP5在心脏发育中的作用，我们培育了一条吗啉斑马鱼来表达REEP5。REEP5在斑马鱼胚胎发育过程中在心肌中表达，与人的REEP5蛋白有很高的同源性(~80%)。我们已经成功地沉默了REEP5在斑马鱼中的表达。这些变种的初步特征显示，胚胎发育受到干扰，心脏形成异常和功能障碍。FISH和组织将通过免疫印迹、免疫荧光、组织学和亮场成像进行评估。

***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.**************