The role of intracellular trafficking pathways in calcium signaling

描述（由申请人提供）：钙信号传导对许多生物过程至关重要。在免疫细胞、骨骼、心脏和骨骼肌、神经元、神经胶质、胰腺和干细胞中起关键作用的NFAT转录因子响应于Ca 2+动员而被激活。NFAT还在发育过程和恶性转化和致癌作用中起关键作用。在非兴奋性细胞中，NFAT转录因子被钙库操纵的Ca 2+进入激活。钙池耗竭导致内质网中的钙传感器STIM 1激活奥赖质膜钙通道。我的赞助商的实验室在HeLa细胞中进行了全基因组RNAi筛选，以确定钙库操作的Ca 2+进入的新成分。该筛选的目的是进一步了解与Ca 2+信号传导相关的复杂途径。此外，由于目前的治疗剂靶向参与Ca 2+信号传导的蛋白质（例如，环孢菌素A，FK 506），来自RNAi筛选的命中物可以作为未来疗法的靶标进行研究。在这里，我建议通过研究我们筛选的四个命中，UEV 3，USP 13，ArfGAP 2和TMP 21，进一步表征Ca 2+信号转导的调节，所有这些都与细胞内运输有关。我选择研究这些命中，因为我以前的经验研究蛋白质和参与贩运的途径。我们推测，贩运机制参与了运动的STIM 1从内部ER膜质膜贴壁ER膜。在本提案的目标1中，我将利用我在细胞生物学和贩运方面的优势，研究UEV 3和USP 13在Ca 2+信号传导中的作用。我将鉴定由UEV 3/USP 13敲低诱导的STIM上的翻译后修饰。我还将鉴定可能与UEV 3和USP 13形成复合物的其他蛋白质，并将研究UEV 3和USP 13与STIM 1和ORAI 1的相关定位。在目标2中，我将用ArfGAP 2和TMP 21进行类似的实验，这两种蛋白质与贩运有关。将研究这两种蛋白质与STIM和奥赖相互作用并影响其功能和定位的能力。在Ai 3中，我将检查来自基因靶向小鼠的细胞中的钙池操纵的Ca 2+进入和生物学功能，所述基因靶向小鼠缺乏或过表达来自筛选的选定的运输命中。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。这些蛋白质的特性将导致阐明新的途径，控制钙信号。

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.