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

描述(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，这些小信号分子反过来调节NRs的调节特性。REV-ERB是一种NR，它与血红素结合，导致NCoR-HDAC1辅阻遏子复合体的招募，同时抑制参与昼夜节律维持和新陈代谢的基因。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？含有一个硫醇-二硫化物氧化还原开关，根据氧化还原平衡调节血红素的结合。这项建议的第一个具体目标是描述纯全长Rev-erb与DNA和辅阻遏子的相互作用。REV-ERB？通过结合其目标基因的启动子序列ROR-RE来实施其转录控制。首先，通过绘制有助于FLRev-erb：heme Soret带(由UV-可见光光谱的去卷积确定)位置的二硫醇：二硫键群体的比率作为环境氧化还原电位的函数来计算硫醇-二硫键开关的中点氧化还原电位，该氧化还原电位将使用还原/氧化的谷胱甘肽对来控制。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之间的相互作用(以及包含氧化还原开关和血红素配体替代的定点变体)。使用FA，我还将确定氧化还原和血红素对FLRev-erb与FSN-NCoR ID1和ID2肽亲和力的影响，这模拟了FLRev-erb与NCoR的结合。其他工作将集中在重组全长或截短形式的NCoR或HDAC1的生产和纯化以及它们与FLRev-erb的相互作用上。用紫外可见光谱和电子顺磁共振研究了气体传递体与FLRev-erb的结构-功能关系，确定了CO、NO或H_2S与FLRev-erb相互作用的Kd，并表征了它们的配位环境。最后，我将确定NO、CO和H_2S对FLRev-erb和ROR-RE/辅阻遏子相互作用的影响。第二个具体目标将集中于表征调节REV-ERB功能的新的和预先存在的合成配体。虚拟筛选技术将用于筛选与REV-ERB？血红素结合口袋相互作用良好的候选者的化学库。候选化合物和先前描述的叔胺类激动剂/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-erb和ROR-RE/辅阻遏子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¿.