Differential binding of NAD+ and NADH allows the transcriptional corepressor carboxyl-terminal binding protein to serve as a metabolic sensor
Differential binding of NAD+ and NADH allows the transcriptional corepressor carboxyl-terminal binding protein to serve as a metabolic sensor
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DOI：
10.1073/pnas.1633591100
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发表时间：
2003-08-05
影响因子：
11.1
通讯作者：
Goodman, RH
中科院分区：
文献类型：
--
作者：
Fjeld, CC;Birdsong, WT;Goodman, RH
羧基末端结合蛋白（CtBP）是一种转录辅抑制因子，最初通过其与腺病毒EIIA相互作用的能力被发现。CtBP- e1a相互作用受烟酰胺腺嘌呤二核苷酸NAD（+）和NADH的调节，这一发现提出了CtBP作为核氧化还原传感器的可能性。该模型要求NAD+和NADH的结合亲和力存在差异，这一点一直存在争议。通过x射线衍射测定CtBP的结构，发现在烟酰胺腺嘌呤二核苷酸结合位点附近有色氨酸残基。我们发现这个色氨酸残基表现出强烈的荧光共振能量转移到结合的NADH。在本报告中，我们利用这些发现来测量CtBP与NADH和NAD（+）的解离常数。利用荧光共振能量转移法测定NADH的亲和力。NADH与蛋白质的结合与NADH荧光强度增强和最大蓝移有关。通过测量NADH在添加NAD（+）时解离时荧光蓝移的损失来估计NAD（+）的亲和力。我们的研究表明，NADH的亲和力比NAD（+）高100倍，这与CtBP作为核氧化还原传感器的功能一致。此外，半最大结合所需的NADH和NAD（+）浓度与它们在核室中的浓度大致相同。这些发现支持了核烟酰胺腺嘌呤二核苷酸的变化可能调节CtBP在细胞分化、发育或转化中的功能。
Carboxyl-terminal binding protein (CtBP) is a transcriptional corepressor originally identified through its ability to interact with adenovirus EIIA. The finding that CtBP-E1A interactions were regulated by the nicotinamide adeninine dinucleotides NAD(+) and NADH raised the possibility that CtBP could serve as a nuclear redox sensor. This model requires differential binding affinities of NAD+ and NADH, which has been controversial. The structure of CtBP determined by x-ray diffraction revealed a tryptophan residue adjacent to the proposed nicotinamide adenine dinucleotide binding site. We find that this tryptophan residue shows strong fluorescence resonance energy transfer to bound NADH. In this report, we take advantage of these findings to measure the dissociation constants for CtBP with NADH and NAD(+). The affinity of NADH was determined by using fluorescence resonance energy transfer. The binding of NADH to protein is associated with an enhanced intensity of NADH fluorescence and a blue shift in its maximum. NAD(+) affinity was estimated by measuring the loss of the fluorescence blue shift as NADH dissociates on addition of NAD(+). Our studies show a >100-fold higher affinity of NADH than NAD(+), consistent with the proposed function of CtBP as a nuclear redox sensor. Moreover, the concentrations of NADH and NAD(+) required for half-maximal binding are approximately the same as their concentrations in the nuclear compartment. These findings support the possibility that changes in nuclear nicotinamide adenine dinucleotides could regulate the functions of CtBP in cell differentiation, development, or transformation.