Redox Modulation of Repression by Rev-erb, a Heme-Binding Nuclear Receptor

Rev-erb（一种血红素结合核受体）对抑制的氧化还原调节

基本信息

批准号：
8455459
负责人：
Eric Lee Carter
金额：
$ 4.92万
依托单位：
UNIVERSITY OF MICHIGAN AT ANN ARBOR
依托单位国家：
美国
项目类别：
财政年份：
2014
资助国家：
美国
起止时间：
2014-02-01 至 2016-06-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8455459
关键词：
Affect Affinity Agonist Algorithms Anions Benchmarking Binding Biological Assay Biological Models Calorimetry Cells Circadian Rhythms Code Complex Computer software Cysteine DNA Data Databases Development Dissociation Disulfides Electron Spin Resonance Spectroscopy Elements Environment Escherichia coli Fluorescein Fluorescence Anisotropy Gases Gel Chromatography Gene Targeting Glutathione Disulfide HDAC1 gene Heme Homeostasis Human ID2 gene Label Lead Length Ligands Maintenance Measures Metabolic Metabolic Diseases Metabolism Methods Monitor Nuclear Hormone Receptors Nuclear Receptors Oligonucleotides Output Oxidation-Reduction Peptides Pharmacologic Substance Population Positioning Attribute Preparation Production Property Protein Binding Proteins Recombinants Repression Signaling Molecule Site Sleep Disorders Spectrum Analysis Structure Structure-Activity Relationship Sulfhydryl Compounds Techniques Testing Thermodynamics Titrations Transcriptional Regulation Variant analytical ultracentrifugation base dithiol gene repression heme a in vivo innovation insight novel promoter public health relevance receptor binding research study screening sedimentation equilibrium small molecule libraries tertiary amine transcription factor virtual

项目摘要

DESCRIPTION (provided by applicant): Nuclear hormone receptors (NRs) are eukaryotic transcription factors that recognize small signaling molecules, which in turn modulate the regulatory properties of NRs. Rev-erb¿ is a NR that binds heme leading to the recruitment of the NCoR-HDAC1 corepressor complex with concomitant repression of genes involved in circadian rhythm maintenance and metabolism. Rev-erb¿-heme binds CO and NO, gaseous signaling molecules involved in diurnal cycling; additionally, Rev-erb¿ contains a thiol-disulfide redox switch that modulates heme-binding in accordance with redox poise. The first specific aim of this proposal is to describe the interactions of pure full-length Rev-erb¿ (FLRev-erb¿) with DNA and corepressors. Rev-erb¿ exerts its transcriptional control by binding to a promoter sequence of its target genes called ROR-RE. First, the mid- point redox potential of the thiol-disulfide switch will be calculated by plotting the ratio of dithiol:disulfide populations contributing to th position of the FLRev-erb¿:heme Soret band (determined by deconvolution of the UV-visible spectrum) as a function of the ambient redox potential that will be controlled using the reduced/oxidized glutathione couple. Next, fluorescence anisotropy (FA) will be utilized to determine how variations in redox poise and heme concentrations affect the interaction between fluorescein (FSN) labeled ROR-RE oligonucleotides and FLRev-erb¿ (and site-directed variants containing substitutions of the redox switch and heme-ligands). Using FA, I will also determine the influences of redox and heme on the affinity of FLRev-erb¿ for FSN-NCoR ID1 and ID2 peptides, which mimic the binding of FLRev-erb¿ to NCoR. Additional efforts will focus on the production and purification of recombinant full-length or truncated forms of NCoR or HDAC1 and their interactions with FLRev-erb¿. The structure-function relationship of gasotransmitters binding to FLRev-erb¿-heme will be explored with UV-visible spectroscopy and electron paramagnetic resonance to determine a Kd for the interaction between CO, NO or H2S and FLRev-erb¿, and to characterize the coordination environment of heme-gas complexes. Lastly, I will determine the effect of NO, CO and H2S on the interaction between FLRev-erb¿ and ROR-RE/corepressors. The second specific aim will focus on the characterization of novel and pre-existing synthetic ligands that modulate Rev-erb¿ function. Virtual screening techniques will be used to screen chemical libraries for candidates that favorably interact with the Rev-erb¿ heme-binding pocket. The thermodynamics of candidate compounds and previously described tertiary amine- based agonists/antagonists binding to FLRev-erb¿ will be measured with ITC and the influence of the ligands on heme-binding will be tested with UV-visible spectroscopy and EPR. Lastly, I will determine the effect of synthetic ligands on the interaction between FLRev-erb¿ and ROR-RE/corepressors. Results obtained during pursuance of these specific aims will lead to a coherent biological model explaining how cellular redox poise and heme control the regulatory output of Rev-erb¿.

描述（由申请人提供）：核激素受体（NR）是识别小信号分子的真核转录因子，进而调节 NR 的调节特性。是一种结合血红素的 NR，导致 NCoR-HDAC1 辅阻遏物复合物的募集，同时抑制参与昼夜节律维持和代谢的基因。 -血红素结合 CO 和 NO，参与昼夜循环的气体信号分子；此外，Rev-erb¿含有硫醇-二硫化物氧化还原开关，可根据氧化还原平衡调节血红素结合。该提案的第一个具体目标是描述纯全长 Rev-erb 的相互作用。 (FLRev-erb¿) 与 DNA 和辅阻遏物。首先，硫醇-二硫键开关的中点氧化还原电位将通过绘制对 th 位置有贡献的二硫醇：二硫键群体的比率来计算。 FLRev-erb¿ ：血红素索雷带（通过紫外-可见光谱的反卷积确定）作为环境氧化还原电位的函数，该电位将使用还原型/氧化型谷胱甘肽对进行控制接下来，将利用荧光各向异性 (FA) 来确定如何变化。氧化还原平衡和血红素浓度影响荧光素 (FSN) 标记的 ROR-RE 寡核苷酸和 FLRev-erb 之间的相互作用¿ （以及包含氧化还原开关和血红素配体取代的定点变体），我还将确定氧化还原和血红素对 FLRev-erb 亲和力的影响。用于 FSN-NCoR ID1 和 ID2 肽，模拟 FLRev-erb 的结合其他工作将集中于 NCoR 或 HDAC1 的重组全长或截短形式的生产和纯化及其与 FLRev-erb 的相互作用。 .与 FLRev-erb 结合的气体递质的结构-功能关系¿ -血红素将通过紫外-可见光谱和电子顺磁共振进行探索，以确定 CO、NO 或 H2S 与 FLRev-erb 之间相互作用的 Kd¿ ，并表征血红素-气体复合物的配位环境。最后，我将确定 NO、CO 和 H2S 对 FLRev-erb 之间相互作用的影响。第二个具体目标将集中于调节 Rev-erb 的新型和现有合成配体的表征。虚拟筛选技术将用于筛选化学库中与 Rev-erb¿候选化合物和之前描述的与 FLRev-erb 结合的叔胺激动剂/拮抗剂的热力学。将使用 ITC 进行测量，并使用紫外可见光谱和 EPR 测试配体对血红素结合的影响。最后，我将确定合成配体对 FLRev-erb 之间相互作用的影响。在追求这些特定目标过程中获得的结果将产生一个连贯的生物学模型，解释细胞氧化还原平衡和血红素如何控制 Rev-erb 的调节输出。。