Study of regulation mechanism for NPAS2 by environmental factors such as CO and NO

神经元PAS结构域蛋白2 （NPAS2）具有两个血红素结合位点，即PASA和PASB结构域，是调节哺乳动物前脑昼夜节律的转录因子。在本研究中，我们分析了血红素结构域的结构与功能之间的关系。分离的PASA的共振拉曼光谱表明，铁态主要由6位低自旋种组成。C170A突变导致可分配给Fe^<3+>-S拉伸的条带急剧减少，表明Cys170是铁血红素的轴向配体。还原形式的共振拉曼光谱主要为6位低自旋型，突变体分析表明，在亚铁血红素中，His119和His171是轴向配体，而不是Cys170，并且在血红素还原过程中发生了从Cys到His的配体置换。另一方面，分离的bHLH-PASA结构域的共振拉曼光谱表明，His119和His171是铁和铁离子的轴向配体。为了表征血红素结构域对功能的作用，我们在NIH3T3细胞中过表达小鼠NPAS2和小鼠BMAL1报告基因，并利用荧光素酶法研究NPAS2血红素结构域突变对mper1和mper2转录表达的影响。具有6位低自旋铁血红素的H138A、H148A和C170A突变体与野生型一样，可以作为mper1和mper2的反激活子。相比之下，H119A和H171A突变体显著降低了转录活性。利用NPAS2铁血红素分离的bHLH-PASA结构域和BMAL1分离的bHLH-PASA- pasb结构域进行体外凝胶转移实验发现，突变体中的H119A和H171A导致了典型E- box (CACGTT) DNA结合活性的丧失。其他突变体如H138A、H148A和C170A具有与野生型相似的显著结合活性。这些结果表明，突变体的转录活性与DNA结合活性具有良好的相关性，表明PAS结构域残基附近的局部构象变化是调控转录活性的原因。进一步的凝胶转移分析表明，NPAS2/BMAL1异源二聚体可以特异性结合mper1中发现的典型E-box序列，但只能弱结合mper2中发现的非典型E-box (CACGTT)，这表明NPAS2对这些基因表达的调节作用不同。少

Neuronal PAS domain protein 2 (NPAS2) with two heme-binding sites, PASA and PASB domains, is a transcription factor regulating circadian rhythm in the mammalian forebrain. In this study, we analyzed the relationship between the structures of heme domains and the functions.1. Resonance Raman spectra of the isolated PASA indicate that the ferric form was predominantly composed of the 6-coordinate low-spin species. The C170A mutation resulted in drastic reduced in a band assignable to Fe^<3+>-S stretching, suggesting that Cys170 is an axial ligand of the ferric heme. The resonance Raman spectra of the reduced form was mainly of 6- coordinate low-spin type, and the mutants analysis revealed that His119 and His171, but not Cys170, are axial ligands in the ferrous heme, and ligand replacement from Cys to His occurs upon heme reduction. On the other hand, resonance Raman spectra of the isolated bHLH-PASA domain suggested that His119 and His171 are axial ligands for both ferric and ferrous com … More plexes.2. To characterize the role of the heme domain on the function, we overexpressed mouse NPAS2 and mouse BMAL1 with a reporter gene in NIH3T3 cells, and investigated the effects of mutations in the heme domain of NPAS2 on transcriptional expression of mper1 and mper2 using luciferase assay. H138A, H148A, and C170A mutants with 6-coordinated low-spin ferric heme demonstrated to act as a transactivator for both mper1 and mper2 like the wlid-type. In contrast, H119A and H171A mutants remarkably reduced transcriptional activity.3. In vitro gel-shift assay using the isolated bHLH-PASA domain of NPAS2 with ferric heme and bHLH-PASA-PASB domain of BMAL1 revealed that H119A and H171A among the mutants resulted in loss of DNA binding activity to the canonical E- box (CACGTT). The other mutants such as H138A, H148A and C170A had significant binding activity similar to that of wild-type. These results indicate that transcriptional activities of the mutants correlated well with the DNA binding activities, suggesting the local conformational changes near these residues of PAS domain is responsible for the regulation of the transcriptional activity.4. Further analysis using gel-shift demonstrated that NPAS2/BMAL1 heterodimer could specifically bind to the canonical E-box sequence found in mper1, but only weakly bind to a non-canonical E-box (CACGTT) found in mper2, suggesting that NPAS2 works differently for regulation of these gene expressions. Less