Functional analysis of the CAMTA Transcription Factors

描述(申请人提供)：我们研究的长期目标是了解细胞如何在不同的生理条件下维持细胞内钙稳态。作为一种多功能的信号分子，钙离子调控着几乎所有类型的细胞的增殖、分化、功能、衰老和凋亡。钙稳态异常可能会对细胞造成损害，并与衰老和许多人类疾病有关，如阿尔茨海默病和脊髓小脑性共济失调。为了避免钙离子的不良影响，细胞内所有的钙离子进入途径都受到严格的控制。G蛋白偶联受体(GPCRs)通过GQ/11蛋白和磷脂酶C(PLC)激活一组普遍表达的钙离子内流通道，称为受体操纵的钙离子/阳离子通道(ROCs)。由于大多数GPCRs和ROC在包括神经元在内的可兴奋细胞中的蛋白水平要高得多，为了维持细胞内的钙稳态，这些细胞需要通过表达更多的调节分子来加强GPCRs/ROC的调控机制。我们先前已经证明，dCAMTA是一种响应钙离子传感器钙调蛋白的转录因子，对于果蝇眼睛中光刺激的GPCR视紫质的快速失活是必不可少的。DCAMTA属于一个新的转录因子家族，称为钙调蛋白结合转录激活因子(CAMTA)。有趣的是，人类的CAMTA，CAMTA1和CAMTA2，都在大脑中高度表达。我们预测，钙调蛋白/CAMTA刺激的基因表达可能以一种长期反馈的方式加强GPCR/ROC介导的神经元内钙内流的控制机制。为了验证这一假设，我们建议使用dCAMTA作为模型，并利用苍蝇光转导级联，这是一个典型的GPCR/PLC级联，已成功地用于识别第一个ROC通道Trp。在这个提议中，我们将1.检验dCAMTA靶基因dFbx14对于视紫红质的快速失活是必不可少的假说；2.检验dFbx14与肌球蛋白III NINAC相互作用以失活视紫红质的假说；3.检验dCAMTA促进钙调蛋白表达以促进视紫红质失活的假说；4.检验dCAMTA缺失导致老年果蝇钙依赖的空泡光感受器变性的假说；5.鉴定dCAMTA和人CAMTA1的核定位序列；6.检验dCAMTA和/或Cam1是人CAMTA1靶基因的假说。公共卫生相关性：钙稳态缺陷与多种人类疾病有关，包括几种神经退行性疾病(阿尔茨海默病和脊髓小脑性共济失调)和几种形式的免疫缺陷。这项研究的长期目标是充分了解细胞内钙稳态是如何在生理和病理条件下维持的，并利用这一知识促进治疗，也许是预防这些人类疾病。这项建议将研究一组新的转录因子如何以反馈方式调节钙稳态。

DESCRIPTION (provided by applicant): The long-term goal of our research is to understand how cells maintain intracellular Ca2+ homeostasis in diverse physiological conditions. As a versatile signaling molecule, Ca2+ regulates the proliferation, differentiation, function, aging, and apoptosis of virtually all types of cells. Abnormal calcium homeostasis may cause damage to the cell, and has been implicated in aging and in numerous human diseases, such as Alzheimer's disease and spinocerebellar ataxia. To avoid undesirable effects of Ca2+, all pathways of Ca2+ entry are tightly controlled in the cell. A group of ubiquitously expressed Ca2+ influx channels, termed receptor-operated Ca2+/cation channels (ROCs), are stimulated by G protein-coupled receptors (GPCRs) through Gq/11 proteins and phospholipase C (PLC). Since most GPCRs and ROCs have much higher protein levels in excitable cells including neurons, to maintain the intracellular Ca2+ homeostasis, these cells need to fortify the regulatory machinery of GPCR/ROC by expressing more regulatory molecules. We have previously demonstrated that dCAMTA, a transcription factor responding to the Ca2+ sensor calmodulin, is indispensable for rapid deactivation of the light-stimulated GPCR rhodopsin in the Drosophila eye. dCAMTA belongs to a new family of transcription factors named calmodulin-binding transcription activators (CAMTAs). Interestingly, both human CAMTAs, CAMTA1 and CAMTA2, are highly expressed in the brain. We predict that the calmodulin/CAMTA-stimulated gene expression may fortify the control machinery of GPCR/ROC-mediated Ca2+ entry in neurons, in a long-term feedback manner. To test this hypothesis, we propose to use dCAMTA as model and to take advantage of the fly phototransduction cascade, a typical GPCR/PLC cascade that has been successfully used for the identification of the first ROC channel TRP. In this proposal, we will 1. Test the hypothesis that the dCAMTA target gene dFbxl4 is indispensable for rapid deactivation of rhodopsin; 2. Test the hypothesis that dFbxl4 interacts with the myosin III NINAC for rhodopsin deactivation; 3. Test the hypothesis that dCAMTA promotes expression of calmodulin to facilitate the deactivation of rhodopsin; 4. Test the hypothesis that loss of dCAMTA leads to Ca2+-dependent, vacuolar photoreceptor degeneration in older flies; 5. identify the nuclear localization sequences of dCAMTA and human CAMTA1; 6. Test the hypothesis that Fbxl4 and/or cam1 are target genes of human CAMTA1. PUBLIC HEALTH RELEVANCE: Defects in calcium homeostasis have been implicated in a variety of human disorders including several neurodegeneration diseases (Alzheimer's disease and spinocerebellar ataxia) and several forms of immunodeficiency The long- term goal of this research is to fully understand how the intracellular Ca2+ homeostasis is maintained in both physiological and pathological conditions and to use this knowledge to facilitate treatment and perhaps prevention of these human diseases. This proposal will study how a new group of transcription factors regulate the Ca2+ homeostasis in a feedback manner.