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

基本信息

批准号：
10341170
负责人：
Jennifer Nhieu
金额：
$ 4.32万
依托单位：
UNIVERSITY OF MINNESOTA
依托单位国家：
美国
项目类别：
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/10341170
关键词：
Address Affect Binding Proteins Biochemical Biological Biological Assay Biological Process CREB1 gene Ca(2+)-Calmodulin Dependent Protein Kinase Calcium Calmodulin Cell Cycle Regulation Cell Line Cell physiology Complex Data Development Disease Dissection Elements Endocrine Extracellular Signal Regulated Kinases Fingerprint Foundations 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 Play Protein Isoforms Regulation Reporter Reporting Retinoic Acid Receptor Retinoids Role 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 design human disease insight interest novel novel therapeutic intervention nutrition quantum reconstitution stem cells structural biology viral rescue

项目摘要

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：全反式视黄酸（atRA）是维生素A的活性代谢产物，通过视黄酸发挥作用。越来越多的研究报告不依赖于 RAR（非规范）。重要的是，基因研究已证实细胞中 atRA 信号传导的活性。视黄酸结合蛋白 1 (Crabp1) 介导这种 atRA 非典型活性，例如快速调节最近，我们发现了干细胞周期控制中的细胞外信号调节激酶（ERK）激活。 Crabp1 的另一个信号传导靶标是钙 (Ca2+)-钙调蛋白依赖性蛋白激酶 II (CaMKII)。该提案中的初步数据支持了一种与多种细胞功能有关的重要激酶。 Crabp1-CaMKII 信号小体在介导维生素 A 作为营养物质的作用中的生理相关性我们的初步数据显示 CaMKII 发生了显着改变。人类遗传数据发现，Crabp1 敲除 (CKO) 小鼠运动神经元的活性显着降低。 Crabp1 在某些运动神经元疾病中表达，进一步支持生理和病理与 Crabp1 的相关性主要假设是 Crabp1 信号体编排了一部小说（RAR- 独立的）atRA 响应机制，严格调节重要细胞内信号传导的激活 CaMKII 和 ERK 等成分。提案）在调节运动神经元活动中发挥作用。我的提案的目标是阐明其机制。为了实现这一目标，我们提出了两个目标：目标 1。利用分子、生物化学和结构生物学精确确定 Crabp1 和 CaMKII 之间的分子关系 atRA 和我们库中的化合物（Crabp1 选择性和 RAR 独立）对的影响。这一分子关系也将得到确定，目标 2 将在功能上验证 Crabb1 调节 CaMKII 的作用。 Ca2+ 调节神经元功能的生理相关细胞环境中的活性。通过使用a）重建的神经细胞系来分子解剖所需的关键元素来完成用于 CaMKII 信号转导和 b) 原代 CKO 运动神经元中 Crabp1 表达的病毒拯救，然后该 Crabp1-CaMKII 通路的功能验证包括 1) CaMKII 的磷酸化。底物 AMPAR、CREB 和突触蛋白 2) 介导的细胞内 Ca2+ 浓度的测量。神经元活动/放电（CaMKII 活动的已知输出）。该提案的意义在于提供第一个。对维生素 A/atRA 调节 CaMKII 活性的新非规范信号传导的分子理解在营养/内分泌方面，该提案在预防某些人类疾病方面发挥着作用。解决了 atRA（一种内分泌和必需营养素）与重要的 Ca2+ 信号传导之间的新交叉问题分子，CaMKII，结果将为人们认识到维生素 A 的复杂性提供深入的见解。信号传导和针对人类疾病的新型类视黄醇疗法的开发。