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

CO、NO等环境因子对NPAS2的调控机制研究

• 批准号: 17570118
• 负责人: SAGAMI Ikuko
• 金额: $ 2.24万
• 依托单位: Kyoto Prefectural University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 2005
• 资助国家: 日本
• 起止时间: 2005 至 2006
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-17570118/
• 关键词: Clock gene; Heme protein; Gas-sensor; Transcription factor; Gel-shift assay; NADPH; PAS protein

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

神经元PAS结构域蛋白2（NPAS 2）具有两个血红素结合位点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的关系.分离的PASA的共振拉曼光谱表明，铁的形式主要由6-配位的低自旋物种。C170 A突变导致Fe^<3+>-S伸缩带的急剧减少，表明Cys 170是铁血红素的轴向配体。还原态的共振拉曼光谱主要为6-配位低自旋型，突变体分析表明亚铁血红素中的轴向配体为His 119和His 171，而非Cys 170，血红素还原时配体发生了从Cys到His的置换.另一方面，分离的bHLH-PASA结构域的共振拉曼光谱表明His 119和His 171是铁和亚铁离子的轴向配体。 ...更多信息 丛。为了表征血红素结构域对功能的作用，我们在NIH 3 T3细胞中过表达小鼠NPAS 2和小鼠BMAL 1与报告基因，并使用荧光素酶测定研究NPAS 2血红素结构域突变对mper 1和mper 2转录表达的影响。H138 A，H148 A，和C170 A突变体与6-协调的低自旋铁血红素被证明作为一个反式激活剂的mper 1和mper 2像野生型。相比之下，H119 A和H171 A突变体的转录活性显著降低.使用分离的NPAS 2的bHLH-PASA结构域与铁血红素和BMAL 1的bHLH-PASA-PASB结构域进行的体外凝胶位移测定显示，突变体中的H119 A和H171 A导致DNA与经典E盒（CACGTT）的结合活性丧失。其他突变体如H138 A、H148 A和C170 A具有与野生型相似的结合活性。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，说明PAS结构域的这些残基附近的局部构象变化是转录活性调节的原因.进一步的凝胶迁移分析表明，NPAS 2/BMAL 1异二聚体可以特异性结合mper 1中的经典E-box序列，但仅与mper 2中的非经典E-box（CACGTT）弱结合，表明NPAS 2对这些基因表达的调控作用不同。少

项目成果

Spectroscopic and DNA-binding characterization of the isolated hemebound basic helix-loop-helix-PAS-A domain of neuronal PAS protein 2(NPAS2), a transcription activator protein asociated with circadian rhythms.

神经元 PAS 蛋白 2 (NPAS2) 的分离血红素结合碱性螺旋-环-螺旋-PAS-A 结构域的光谱和 DNA 结合特征，NPAS2 是一种与昼夜节律相关的转录激活蛋白。

• 发表时间: 2006
• 期刊: FEBS J. 273・11
• 作者: Mukaiyama Y;Uchida T;Sato E;Sasaki A;Sato Y;Igarashi J;Kurokawa H;Sagami I;Kitagawa T;Shimizu T.
• 通讯作者: Shimizu T.

Spectroscopic characterization of the isolated heme-bound PAS-B domain of neuronal PAS domain protein 2 associated with circadian rhythms

• DOI: 10.1111/j.1742-4658.2005.04828.x
• 发表时间: 2005-08-01
• 期刊: FEBS JOURNAL
• 影响因子: 5.4
• 作者: Koudo, R;Kurokawa, H;Shimizu, T
• 通讯作者: Shimizu, T

Characterization of heme-regulated dIF2alpha kinase : roles of the N-terminal domain in the oligomeric state, heme binding, catalysis, and inhibition.

血红素调节的 dIF2α 激酶的表征：N 末端结构域在寡聚状态、血红素结合、催化和抑制中的作用。

• 发表时间: 2006
• 期刊: Biochemistry 15・32
• 作者: Miksanova M;Igarashi J;Minami M;Sagami I;Yamauchi S;Kurokawa H;Shimizu T
• 通讯作者: Shimizu T

Spectroscopic and DNA-binding characterization of the isolated heme-bound basic helix-loop-helix-PAS-A domain of neuronal PAS protein 2 (NPAS2), a transcription activator protein associated with circadian rhythms

• DOI: 10.1111/j.1742-4658.2006.05259.x
• 发表时间: 2006-06-01
• 期刊: FEBS JOURNAL
• 影响因子: 5.4
• 作者: Mukaiyama, Yuji;Uchida, Takeshi;Shimizu, Toru
• 通讯作者: Shimizu, Toru

DOSEc, a Heme-Regulated Phosphodiesterase, Plays an Important Role in the Regulation of the Cyclic AMP Level in Escherichia coli

• DOI: 10.1128/jb.187.19.6678-6682.2005
• 发表时间: 2005-10
• 期刊: Journal of Bacteriology
• 影响因子: 3.2
• 作者: Tokiko Yoshimura-Suzuki;I. Sagami;N. Yokota;H. Kurokawa;Toru Shimizu