Functional analysis of the CAMTA Transcription Factors

描述（由申请人提供）：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Ca2+稳态。作为一种多用途的信号分子，Ca2+调节几乎所有类型细胞的增殖、分化、功能、衰老和凋亡。异常的钙稳态可能导致细胞损伤，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Ca2+的不良影响，Ca2+进入细胞的所有途径都受到严格控制。G蛋白偶联受体（gpcr）通过Gq/11蛋白和磷脂酶C （PLC）刺激一组无处不在表达的Ca2+内流通道，称为受体操作的Ca2+/阳离子通道（ROCs）。由于大多数GPCR和ROC在包括神经元在内的可兴奋细胞中具有更高的蛋白水平，为了维持细胞内Ca2+稳态，这些细胞需要通过表达更多的调节分子来加强GPCR/ROC的调节机制。我们之前已经证明，dCAMTA是一种响应Ca2+传感器钙调蛋白的转录因子，对于果蝇眼睛中光刺激的GPCR视紫红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，称为钙调素结合转录激活因子（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，钙调素/ camta刺激的基因表达可能会以长期反馈的方式加强GPCR/ roc介导的Ca2+进入神经元的控制机制。为了验证这一假设，我们建议使用dCAMTA作为模型，并利用苍蝇光导级联，这是一种典型的GPCR/PLC级联，已成功用于鉴定第一个ROC通道TRP。在本提案中，我们将1。验证dCAMTA靶基因dfbx14在视紫红质快速失活中不可或缺的假设；2. 验证dfbx14与肌球蛋白III NINAC相互作用导致视紫红质失活的假设；3. 验证dCAMTA促进钙调蛋白表达促进视紫红质失活的假说；4. 验证dCAMTA缺失导致老年果蝇Ca2+依赖性空泡光感受器退化的假设；5. 鉴定dCAMTA和人CAMTA1的核定位序列；6. 验证fbx14和/或cam1是人类CAMTA1靶基因的假设。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。本研究的长期目标是充分了解细胞内Ca2+稳态是如何在生理和病理条件下维持的，并利用这些知识促进治疗和预防这些人类疾病。本研究将研究一组新的转录因子如何以反馈的方式调节Ca2+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.