Non-canonical signaling of retinoic acid by Cellular Retinoic Acid Binding Protein 1

细胞视黄酸结合蛋白 1 的非典型视黄酸信号传导

• 批准号: 10553097
• 负责人: Jennifer Nhieu
• 金额: $ 1.36万
• 依托单位: UNIVERSITY OF MINNESOTA
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-03-01 至 2023-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10553097
• 关键词: Address; Affect; Binding Proteins; Biochemical; Biological; Biological Assay; Biological Process; CREB1 gene; Ca(2+)-Calmodulin Dependent Protein Kinase; Calcium; Calcium Signaling; Calmodulin; Calmodulin 1; Cell Cycle Regulation; Cell Line; Cell physiology; Complex; Data; Development; Disease; Dissection; Elements; Endocrine; Extracellular Signal Regulated Kinases; Fingerprint; Gene Expression; Genes; Genetic Models; Genetic study; Goals; Human Genetics; Immunohistochemistry; In Vitro; Knock-out; Knockout Mice; Knowledge; Libraries; Ligand Binding; Measurement; Measures; Mediating; Mitogens; Molecular; Motor Neuron Disease; Motor Neurons; Mutagenesis; Neurons; Nuclear Magnetic Resonance; Nutrient; Output; Pathologic; Pathway interactions; Phosphorylation; Phosphotransferases; Physiological; Protein Isoforms; Regulation; Reporter; Reporting; Retinoic Acid Receptor; Retinoids; Signal Induction; Signal Pathway; Signal Transduction; Signaling Molecule; Surface Plasmon Resonance; Synapsins; Therapeutic; Toxic effect; Transducers; Tretinoin; Validation; Vitamin A; Western Blotting; biological systems; calmodulin-dependent protein kinase II; cellular retinoic acid binding protein; cellular targeting; design; human disease; insight; interest; novel; novel therapeutic intervention; nutrition; quantum; reconstitution; stem cells; structural biology; viral rescue

Abstract: All-trans retinoic acid (atRA) is an active metabolite of vitamin A that acts through retinoic acid receptors (RARs) to regulate gene expression. Increasing studies report RAR-independent (non-canonical) activity of atRA signaling in various biological systems. Importantly, genetic studies have established that cellular retinoic acid binding protein 1 (Crabp1) mediates this atRA non-canonical activity, such as rapid modulation of extracellular signal–regulated kinase (ERK) activation in stem cell cycle control. Recently, we have identified another signaling target of Crabp1, calcium (Ca2+)-calmodulin dependent protein kinase II (CaMKII). CaMKII is an important kinase implicated in numerous cellular functions. Preliminary data in this proposal support the physiological relevance of the Crabp1-CaMKII signalsome in mediating the action of vitamin A as a nutrient and an endocrine that modulates cellular signaling capacity. Our preliminary data reveal dramatically altered CaMKII activity in Crabp1 knockout (CKO) mouse motor neurons. Human genetic data identified significantly reduced Crabp1 expression in certain motor neuron diseases, further supporting the physiological and pathological relevance for Crabp1. The principal hypothesis is that the Crabp1-signalsome orchestrates a novel (RAR- independent) atRA-responsive mechanism that tightly regulates activation of important intracellular signaling components such as CaMKII and ERK. I hypothesize that this Crabp1-CaMKII signalsome (the focus of this proposal) plays a role in modulating motor neuron activity. The goal of my proposal is to elucidate the mechanism of this Crabp1-CamKII signalsome. To achieve this goal, two aims are proposed. Aim 1 will characterize the exact molecular relationship between Crabp1 and CaMKII using molecular, biochemical, and structural biology approaches. The effect of atRA and compounds from our library (Crabp1-selective and RAR-independent) on this molecular relationship will also be determined. Aim 2 will functionally validate Crabp1 in regulating CaMKII activity in a physiologically relevant cellular context of Ca2+-regulated neuronal functions. This will be accomplished by employing a) reconstituted neuron cell lines to molecularly dissect critical elements required for CaMKII signaling and b) viral rescue of Crabp1 expression in primary CKO motor neurons, followed by functional validation of this Crabp1-CaMKII pathway. Functional assays include 1) phosphorylation of CaMKII substrates AMPAR, CREB, and synapsin. 2) Measurement of intracellular Ca2+ concentrations that mediate neuron activity/firing (a known output of CaMKII activity). The significance of this proposal is to provide the first molecular understanding of a new non-canonical signaling of vitamin A/atRA that modulates CaMKII activity and plays a role in protecting against certain human diseases. In a nutrition/endocrine context, this proposal addresses a new cross-talk between atRA (an endocrine and essential nutrient) and an important Ca2+ signaling molecule, CaMKII. The results will provide insight on the increasingly recognized complexity in vitamin A signaling and the development of novel retinoid therapeutics for human disease.

摘要：全反式维甲酸（atRA）是维生素A的活性代谢产物，通过维甲酸发挥作用 RARs（RARs）是调节基因表达的关键。越来越多的研究报告RAR无关（非规范） atRA信号在各种生物系统中的活性。重要的是，遗传学研究已经证实， 视黄酸结合蛋白1（Crabp 1）介导这种atRA非典型活性，例如快速调节 细胞外信号调节激酶（ERK）在干细胞周期调控中的作用。最近，我们发现 Crabp 1的另一个信号靶点是钙（Ca 2+）-钙调蛋白依赖性蛋白激酶II（CaMKII）。CaMKII是 一种参与许多细胞功能的重要激酶。本提案中的初步数据支持 Crabp 1-CaMKII信号体在介导维生素A作为营养物的作用中的生理相关性， 调节细胞信号能力的内分泌。我们的初步数据显示， Crabp 1敲除（CKO）小鼠运动神经元中的活性。人类基因数据识别大幅减少 Crabp 1在某些运动神经元疾病中的表达，进一步支持了运动神经元疾病的生理和病理学机制。 与Crabp 1相关。主要的假设是Crabp 1信号体协调了一种新的（RAR-1）。 非依赖性）atRA反应机制，紧密调节重要的细胞内信号传导的激活 例如CaMKII和ERK。我假设这个Crabp 1-CaMKII信号（这一点的焦点） 建议）在调节运动神经元活动中起作用。我的提案的目的是阐明 这个Crabp 1-CamKII信号。为实现这一目标，提出了两个目标。目标1将描述 使用分子、生物化学和结构生物学方法研究Crabp 1和CaMK II之间的确切分子关系 接近。atRA和来自我们库的化合物（Crabp 1选择性和RAR非依赖性）对 也将确定这种分子关系。目的2将在功能上验证Crabp 1在调节CaMKII中的作用 活性在生理相关的细胞环境中的Ca 2+调节的神经元功能。这将是 通过使用a）重建的神经元细胞系来分子解剖所需的关键元件来完成 用于CaMK II信号传导和B）病毒拯救原代CKO运动神经元中的Crabp 1表达，然后 Crabp 1-CaMKII通路的功能验证。功能测定包括1）CaMKII的磷酸化 底物AMPAR、CREB和突触蛋白。2)测量细胞内Ca 2+浓度，介导 神经元活动/放电（CaMKII活动的已知输出）。这项建议的意义在于提供了第一个 对维生素A/atRA调节CaMKII活性的新非经典信号传导的分子理解， 在预防某些人类疾病方面起着重要作用。在营养/内分泌方面， 解决了atRA（一种内分泌和必需营养素）与一种重要的Ca 2+信号传导之间的新的交叉作用 分子，CaMKII。这些结果将为人们越来越认识到的维生素A的复杂性提供见解 信号传导和开发用于人类疾病的新型类维生素A治疗剂。