Regulators of Ca+ NFAT siginaling a global RNAi screen

Ca NFAT 监管机构发出全球 RNAi 筛查信号

• 批准号: 7114360
• 负责人: Anjana Rao
• 金额: $ 42.68万
• 依托单位: IMMUNE DISEASE INSTITUTE, INC.
• 依托单位国家: 美国
• 财政年份: 2005
• 资助国家: 美国
• 起止时间: 2005-09-01 至 2009-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7114360
• 关键词: B lymphocyte; Drosophilidae; RNA interference; T lymphocyte; bioinformatics; biological signal transduction; biotechnology; calcineurin; calcium flux; chimeric proteins; double stranded RNA; genetic screening; genetically modified animals; green fluorescent proteins; ionomycin; laboratory mouse; mast cell; nuclear transport; thapsigargin; tissue /cell culture; transcription factor

DESCRIPTION (provided by applicant): In lymphocytes and mast cells, stimulation through antigen or Fc receptors leads to depletion of intracellular Ca2+ stores and subsequent Ca2+ influx through specialized Ca2+-release-activated Ca2+ (CRAC) channels in the plasma membrane. The increase in intracellular free Ca2+ ([Ca2+],) levels activate the phosphatase calcineurin, the upstream regulator of the transcription factor NFAT. We describe a strategy, based on global RNAi screening in Drosophila, to identify genes whose products influence [Ca2+ levels, calcineurin activation or localization of NFAT. RNAi in Drosophila cells is much more efficient than in mammalian cells, providing a powerful method to test individually the effects of specific disruption of all genes in the Drosophila genome. We have completed the first phase of this screen, identifying dsRNAs that cause nuclear localization of an NFAT-GFP fusion protein in resting Drosophila cells and so normally function to retain NFAT in the cytoplasm, for instance by maintaining [Ca2+]i and calcineurin activity at low levels in resting cells or counteracting the nuclear shuttling of NFAT. In Aim 1, we will continue analyzing the regulators identified in this screen. In Aim 2, we will perform a new screen to identify proteins that increase [Ca2+]| levels and calcineurin activity or promote the nuclear translocation of NFAT, by screening for dsRNAs that prevent NFAT nuclear import in Drosophila cells stimulated with thapsigargin which causes depletion of Ca2+ stores. In Aim 3, we will assess the involvement of specific candidates previously implicated in Ca2+-calcineurin-NFAT signalling, which may not have shown up in the screens because of redundancy or effects on cell viability or cytoskeletal function. Once regulators that function in Drosophila have been validated, we will test the functions of the mammalian homologues. These studies are expected to provide a comprehensive picture of the signalling networks that modulate [Ca2+]j levels, calcineurin activity and NFAT subcellular localization in Drosophila and mammalian cells. The studies are important because the Ca2+-calcineurin-NFAT signalling pathway has a critical role in many developmental and transcriptional programs, including T cell activation and differentiation, osteoclast differentiation, cardiac valve development, slow-twitch skeletal muscle fiber differentiation, and neuronal and vascular patterning. NFAT is also implicated in many pathological processes, including transplant rejection, inflammation, osteoporosis, myocardial hypertrophy, allergy and autoimmune disease.

描述（由申请人提供）：在淋巴细胞和肥大细胞中，通过抗原或FC受体刺激会导致细胞内Ca2+储存量以及随后通过专用Ca2+释放酶激活的CA2+（CRAC（CRAC）的CA2+涌入的Ca2+涌入）耗竭。细胞内游离Ca2+（[Ca2+]，）水平的增加激活了磷酸酶钙调蛋白，这是转录因子NFAT的上游调节剂。我们描述了一种基于果蝇中全局RNAi筛查的策略，以识别其影响[Ca2+水平，钙调神经磷酸酶激活或NFAT定位的基因。果蝇细胞中的RNAi比在哺乳动物细胞中的效率要高得多，该方法提供了一种强大的方法，可以单独测试果蝇基因组中所有基因的特异性破坏的影响。我们已经完成了此屏幕的第一阶段，鉴定了在静止的果蝇细胞中引起NFAT-GFP融合蛋白核定位的DSRNA，因此通常可以通过维持[Ca2+] I和calcineurin活性在静止的细胞中的核酸含量或核能构成NFAT的核能中的低水平来保留NFAT。在AIM 1中，我们将继续分析此屏幕中确定的调节器。在AIM 2中，我们将执行一个新的屏幕，以识别增加[Ca2+] |的蛋白质。通过筛选DSRNA，可以防止用Thapsigargin刺激的果蝇细胞中NFAT核进口的DSRNA，从而促进NFAT的核易位，或促进NFAT的核易位，从而导致Ca2+储存的耗竭。在AIM 3中，我们将评估先前与Ca2+-Calcineurin-NFAT信号相关的特定候选者的参与，由于对细胞的纤维纤维或细胞骨架功能的影响，可能没有出现在筛选中。一旦在果蝇中发挥作用的调节剂已得到验证，我们将测试哺乳动物同源物的功能。预计这些研究将为果蝇和哺乳动物细胞中调节[Ca2+] J水平，钙调蛋白活性和NFAT亚细胞定位的信号网络提供全面的了解。研究很重要，因为Ca2+ - 钙化蛋白-NFAT信号通路在许多发育和转录程序中具有至关重要的作用，包括T细胞激活和分化，破骨细胞分化，心脏瓣膜发育，慢速扭转型骨骼肌纤维纤维纤维分化，以及神经元和血管模型。 NFAT还与许多病理过程有关，包括移植排斥，炎症，骨质疏松症，心肌肥大，过敏和自身免疫性疾病。