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

描述（由申请人提供）：核激素受体（NRs）是识别小信号分子的真核转录因子，其反过来调节核激素受体的调节特性。Rev-erb¿是一种NR，它结合血红素，导致NCoR-HDAC1辅抑制因子复合物的募集，同时抑制参与昼夜节律维持和代谢的基因。Rev-erb¿-血红素结合参与昼夜循环的气体信号分子CO和NO；此外，Rev-erb¿含有一个硫醇二硫氧化还原开关，根据氧化还原平衡调节血红素结合。本提案的第一个具体目的是描述纯全长Rev-erb¿（FLRev-erb¿）与DNA和辅阻遏子的相互作用。rev - erbb¿通过结合其靶基因的启动子序列（称为ROR-RE）来发挥转录控制作用。首先，硫醇-二硫开关的中点氧化还原电位将通过绘制对FLRev-erb¿：血红素Soret波段有贡献的二硫醇：二硫族的比例（由紫外-可见光谱的反褶积确定）作为环境氧化还原电位的函数来计算，环境氧化还原电位将由还原/氧化谷胱甘肽对控制。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位点定向变体）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¿对FSN-NCoR的ID1和ID2肽亲和力的影响，这两个肽模拟了FLRev-erb¿与NCoR的结合。进一步的工作将集中在NCoR或HDAC1的全长或截短形式的重组和纯化及其与flrev - erbb¿的相互作用上。利用紫外可见光谱学和电子顺磁共振技术研究气体递质与FLRev-erb -heme结合的结构-功能关系，确定CO、NO或H2S与FLRev-erb -¿相互作用的Kd值，表征血红素-气体配合物的配位环境。最后，我将确定NO， CO和H2S对flrev - erbb¿和ROR-RE/辅抑制因子相互作用的影响。第二个具体目标将集中在表征新的和预先存在的合成配体，调节Rev-erb¿功能。虚拟筛选技术将用于筛选候选化学文库，以与rev - erbb¿血红素结合口袋有利地相互作用。候选化合物和先前描述的叔胺基激动剂/拮抗剂与FLRev-erb¿结合的热力学将用ITC测量，配体对血红素结合的影响将用紫外可见光谱和EPR测试。最后，我将确定合成配体对flrev - erbb¿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期间获得的结果将导致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。

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¿.