The role of intracellular trafficking pathways in calcium signaling

描述（由申请人提供）：钙信号在许多生物过程中是必不可少的。NFAT转录因子在免疫细胞、骨骼、心脏和骨骼肌、神经元、胶质细胞、胰腺和干细胞中发挥关键作用，在Ca2+动员的反应中被激活。NFAT在发育过程、恶性转化和癌变中也起着至关重要的作用。在不可兴奋的细胞中，NFAT转录因子被存储操作的Ca2+进入激活。存储损耗导致ORAI质膜Ca2+通道被STIM1激活，STIM1是内质网中的Ca2+传感器。我的保荐人的实验室在HeLa细胞中进行了全基因组RNAi筛选，以识别储存操作的Ca2+进入的新成分。该筛选的目的是进一步了解与Ca2+信号相关的复杂途径。此外，由于目前的治疗方法靶向参与Ca2+信号传导的蛋白质（例如，环孢素A， FK506）， RNAi筛选的命中可以作为未来治疗的靶点进行研究。在这里，我建议进一步表征Ca2+信号的调控，通过研究我们的筛选，UEV3, USP13, ArfGAP2，和TMP21，所有这些都涉及细胞内运输。我之所以选择研究这些撞击，是因为我之前有研究贩运蛋白质和途径的经验。我们假设转运机制参与了STIM1从内质网膜到质膜对应的内质网膜的运动。在本提案的目标1中，我将通过研究UEV3和USP13在Ca2+信号传导中的作用，利用我在细胞生物学和运输方面的优势。我将确定由UEV3/USP13敲低诱导的STIM上的翻译后修饰。我还将鉴定可能与UEV3和USP13形成复合物的其他蛋白质，并将检查UEV3和USP13与STIM1和ORAI1的定位。在Aim 2中，我将对ArfGAP2和TMP21进行类似的实验，这两种蛋白质与贩运有关。这两种蛋白将被研究它们与STIM和ORAI相互作用并影响其功能和定位的能力。在Ai 3中，我将研究来自缺乏或过度表达筛选贩运命中的基因靶向小鼠的细胞中的储存操作Ca2+进入和生物学功能。这些实验将为我提供学习与小鼠遗传模型和原代细胞类型研究相关的新技能和技术的机会。这些蛋白的表征将导致新的途径，控制钙信号的阐明。

DESCRIPTION (provided by applicant): Calcium signaling is essential for numerous biological processes. The NFAT transcription factors, which play key roles in immune cells, bone, heart and skeletal muscle, neurons, glia, pancreas and stem cells, are activated in response to Ca2+ mobilization. NFAT also plays crucial roles in developmental processes and malignant transformation and carcinogenesis. In non-excitable cells, NFAT transcription factors are activated by store-operated Ca2+ entry. Store depletion results in the activation of the ORAI plasma membrane Ca2+ channels by STIM1, a Ca2+ sensor in the endoplasmic reticulum. My sponsor's lab performed a genome-wide RNAi screen in HeLa cells to identify novel components of store-operated Ca2+ entry. The goal of this screen was to further understand the complex pathways associated with Ca2+ signaling. In addition, because current therapeutics target proteins involved in Ca2+ signaling (e.g., cyclosporin A, FK506), the hits from the RNAi screen could be studied as targets of future therapies. Here I propose to further characterize the regulation of Ca2+ signaling by studying four hits from our screen, UEV3, USP13, ArfGAP2, and TMP21, all of which have been implicated in intracellular trafficking. I chose to study these hits because I have previous experience studying proteins and pathways involved in trafficking. We hypothesize that trafficking mechanisms are involved in the movement of STIM1 from internal ER membranes to plasma membrane-apposed ER membranes. In Aim 1 of this proposal, I will capitalize on my strengths in cellular biology and trafficking by investigating the role of UEV3 and USP13 in Ca2+ signaling. I will identify post-translational modifications on STIM that are induced by UEV3/USP13 knockdown. I will also identify other proteins that might form a complex with UEV3 and USP13, and I will examine the localization of UEV3 and USP13 in relation to STIM1 and ORAI1. In Aim 2, I will perform similar experiments with ArfGAP2 and TMP21, two proteins that have been implicated in trafficking. Both proteins will be investigated for their ability to interact with and affect the function and localization of STIM and ORAI. In Ai 3, I will examine store- operated Ca2+ entry and biological function in cells from gene-targeted mice lacking or overexpressing selected trafficking hits from the screen. These experiments will provide me the opportunity to learn new skills and techniques associated with mouse genetic models and the study of primary cell types. The characterization of these proteins will lead to the elucidation of novel pathways that control calcium signaling.