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Abstract：
NPAS2 is a transcription factor that regulates mammalian circadian rhythms. It has been suggested that NPAS2 DNA-binding activity is regulated by the intracellular redox state of NAD(P)H, although the mechanism remains unclear. To investigate the NAD(P)H interaction site of murine NPAS2, we performed electrophoretic mobility shift assays using several truncation mutants of the NPAS2 bHLH domain. Among the mutants, NPAS2 containing the N-terminal 61 residues formed a heterodimer with BMAL1 to bind DNA, and NAD(P)H enhanced the binding activity, while NAD(P)H inhibited the DNA-binding activity of the BMAL1 homodimer in a dose-dependent manner. NAD(P)H derivatives such as 2\',5\'-ADP, nicotinamide, nicotinic acid and nicotinic acid adenine dinucleotide (NAAD) did not affect the DNA-binding activity. Interestingly, NAD(P)(+), previously reported as an inhibitor, did not affect NPAS2 binding activity in the presence or absence of NAD(P)H in our system. These results suggest that NPAS2 DNA-binding activity is specifically enhanced by NAD(P)H independently of NAD(P)(+) and that the N-terminal 1-61 amino acids of NPAS2 are sufficient to sense NAD(P)H.
摘要：
NPAS2是调节哺乳动物昼夜节律的转录因子。有人提出NPAS2DNA结合活性受NAD（P）H的细胞内氧化还原状态调节，尽管机制尚不清楚。为了研究鼠NPAS2的NAD（P）H相互作用位点，我们使用NPAS2bHLH结构域的几种截短突变体进行了电泳迁移率变化测定。在变种人中,含有N末端61个残基的NPAS2与BMAL1形成异二聚体以结合DNA，NAD（P）H增强了结合活性，而NAD（P）H以剂量依赖性方式抑制BMAL1同二聚体的DNA结合活性。NAD(P)H衍生物，如2'，5\'-ADP,烟酰胺,烟酸和烟酸腺嘌呤二核苷酸（NAAD）不影响DNA结合活性。有趣的是,NAD(P)(+),以前报道为抑制剂，在我们的系统中存在或不存在NAD（P）H时，不影响NPAS2结合活性。这些结果表明，NPAS2DNA结合活性被NAD（P）H特异性增强，而与NAD（P）（+）无关，并且NPAS2的N端1-61个氨基酸足以使NAD（P）H。
