Affordable Access

deepdyve-link deepdyve-link
Publisher Website

Changes in pH and NADPH regulate the DNA binding activity of neuronal PAS domain protein 2, a mammalian circadian transcription factor.

Authors
  • Yoshii, Katsuhiro1
  • Tajima, Fumihisa
  • Ishijima, Sumio
  • Sagami, Ikuko
  • 1 Graduate School of Life and Environmental Science, Kyoto Prefectural University , Shimogamo, Sakyo-ku, Kyoto 606-8522, Japan. , (Japan)
Type
Published Article
Journal
Biochemistry
Publisher
American Chemical Society
Publication Date
Jan 20, 2015
Volume
54
Issue
2
Pages
250–259
Identifiers
DOI: 10.1021/bi5008518
PMID: 25526362
Source
Medline
License
Unknown

Abstract

Neuronal PAS domain protein 2 (NPAS2) is a core clock transcription factor that forms a heterodimer with BMAL1 to bind the E-box in the promoter of clock genes and is regulated by various environmental stimuli such as heme, carbon monoxide, and NAD(P)H. In this study, we investigated the effects of pH and NADPH on the DNA binding activity of NPAS2. In an electrophoretic mobility shift (EMS) assay, the pH of the reaction mixture affected the DNA binding activity of the NPAS2/BMAL1 heterodimer but not that of the BMAL1/BMAL1 homodimer. A change in pH from 7.0 to 7.5 resulted in a 1.7-fold increase in activity in the absence of NADPH, and NADPH additively enhanced the activity up to 2.7-fold at pH 7.5. The experiments using truncated mutants revealed that N-terminal amino acids 1-61 of NPAS2 were sufficient to sense the change in both pH and NADPH. We further analyzed the kinetics of formation and DNA binding of the NPAS2/BMAL1 heterodimer at various pH values. In the absence of NADPH, a change in pH from 6.5 to 8.0 decreased the KD(app) value of the E-box from 125 to 22 nM, with an 8-fold increase in the maximal level of DNA binding for the NPAS2/BMAL1 heterodimer. The addition of NADPH resulted in a further decrease in KD(app) to 9 nM at pH 8.0. Furthermore, NPAS2-dependent transcriptional activity in a luciferase assay using NIH3T3 cells also increased with the pH of the culture medium. These results suggest that NPAS2 has a role as a pH and metabolite sensor in regulating circadian rhythms.

Report this publication

Statistics

Seen <100 times