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

• 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
• 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

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，小信号分子进而调节NR的调节特性。Rev-erb是一种结合血红素的NR，导致NCoR-HDAC 1辅阻遏物复合物的募集，伴随着参与昼夜节律维持和代谢的基因的阻遏。Rev-erb- 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，根据氧化还原平衡调节血红素结合。该建议的第一个具体目标是描述纯全长Rev-erb（FLRev-erb）与DNA和辅阻遏物的相互作用。Rev-erb通过与其靶基因ROR-RE的启动子序列结合来发挥其转录控制作用。首先，将通过绘制对FLRev-erb：血红素Soret带的位置有贡献的二硫醇：二硫化物群体的比率（通过UV-可见光谱的去卷积确定）作为将使用还原/氧化谷胱甘肽对控制的环境氧化还原电位的函数来计算硫醇-二硫化物开关的中点氧化还原电位。接下来，将利用荧光各向异性（FA）来确定氧化还原平衡和血红素浓度的变化如何影响荧光素（FSN）标记的ROR-RE寡核苷酸和FLRev-erb？（以及含有氧化还原开关和血红素配体取代的定点变体）之间的相互作用。使用FA，我还将确定氧化还原和血红素对FLRev-erb？与FSN-NCoR ID 1和ID 2肽的亲和力的影响，这模拟了FLRev-erb？与NCoR的结合。额外的努力将集中在生产和纯化重组全长或截短形式的NCoR或HDAC 1及其与FLRev-erb？的相互作用。将利用紫外可见光谱和电子顺磁共振来探索与FLRev-erb ²-血红素结合的气体递质的结构-功能关系，以确定CO、NO或H2S与FLRev-erb ²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？和ROR-RE/corepressors之间相互作用的影响。第二个具体的目标将集中在新的和预先存在的合成配体，调节Rev-erb的功能的表征。虚拟筛选技术将被用来筛选化学库的候选人，有利地与Rev-erb <$血红素结合口袋。候选化合物和先前描述的基于叔胺的激动剂/拮抗剂与FLRev-erb？结合的热力学将用ITC测量，并且配体对血红素结合的影响将用UV-可见光谱和EPR测试。最后，我将确定合成配体对FLRev-erb？和ROR-RE/辅阻遏物之间相互作用的影响。在追求这些特定目标的过程中获得的结果将导致一个连贯的生物模型，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。