2006 Fiscal Year Final Research Report Summary
Study of regulation mechanism for NPAS2 by environmental factors such as CO and NO
| Project/Area Number |
17570118
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| Research Category |
Grant-in-Aid for Scientific Research (C)
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| Allocation Type | Single-year Grants |
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| Section | 一般 |
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| Research Field |
Functional biochemistry
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| Research Institution | Kyoto Prefectural University |
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Principal Investigator |
SAGAMI Ikuko Kyoto Prefectural University, Professor, 農学研究科, 教授 (10143033)
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| Project Period (FY) |
2005 – 2006
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| Keywords | Clock gene / Heme protein / Gas-sensor / Transcription factor / Gel-shift assay / NADPH / PAS protein |
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| Research Abstract |
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
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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
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Research Products
(8 results)
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[Journal Article] 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.2006
Author(s)
Mukaiyama Y, Uchida T, Sato E, Sasaki A, Sato Y, Igarashi J, Kurokawa H, Sagami I, Kitagawa T, Shimizu T
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Journal Title
FEBS J. 273・11
Pages: 2528-2539
Description
「研究成果報告書概要(和文)」より
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[Journal Article] 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.2006
Author(s)
Mukaiyama Y, Uchida T, Sato E, Sasaki A, Sato Y, Igarashi J, Kurokawa H, Sagami I, Kitagawa T, Shimizu T.
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Journal Title
FEBS J. 273
Pages: 2528-2539
Description
「研究成果報告書概要(欧文)」より
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