The sequence-specific DNA binding of NF-κB is reversibly regulated by the automodification reaction of poly (ADP-ribose) polymerase 1
The sequence-specific DNA binding of NF-κB is reversibly regulated by the automodification reaction of poly (ADP-ribose) polymerase 1
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DOI：
10.1074/jbc.m104666200
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发表时间：
2001-12-14
影响因子：
4.8
通讯作者：
Alvarez-Gonzalez, R
中科院分区：
文献类型：
--
作者：
Chang, WJ;Alvarez-Gonzalez, R
最近的研究表明，聚（ADP-核糖）聚合酶-1 (PARP-1) 催化的蛋白质结合 ADP-核糖聚合物的合成可调节真核基因表达，包括 NF-kappaB 依赖性途径。在这里，我们报告了 PARP-1 激活 NF-kappaB 与其寡脱氧核苷酸序列特异性结合的分子机制。我们在存在或不存在 β NAD(+) 的情况下在体外共孵育纯重组人 PARP-1 和 NF-kappaB 的 p50 亚基 (NF-kappaB-p50)。电泳迁移率变动分析表明，当 PARP-1 存在时，NF-kappaB-p50 DNA 结合依赖于 beta NAD(+) 的存在。在缺乏 beta NAD(+) 的情况下，NF-kappaB-p50 与 DNA 的结合效率不高。事实上，在 3-氨基苯甲酰胺 (3-AB) 存在的情况下，结合也不是有效的。因此，我们得出结论，NF-kappaB-p50 DNA 结合是蛋白质-聚（ADP-核糖基化）依赖性的。免疫共沉淀和免疫印迹分析表明，在不存在 beta NAD（+）的情况下，PARP-1 与 NF-kappaB-p50 物理上具有高特异性相互作用。由于 NF-kappaB-p50 不是聚（ADP-核糖基化）的有效共价靶标，因此我们的结果与结论是，PARP-1 催化的自聚（ADP-核糖基）化反应通过抑制 NF-kappaB-p50 和 PARP-1 之间的特异性蛋白质-蛋白质相互作用，促进 NF-kappaB-p50 与其 DNA 的结合。实验中，在氧化损伤之前将 HeLa 细胞与 3-AB 预孵育，也会强烈抑制体内 NF-κB 的激活。
Recent studies suggest that the synthesis of protein-bound ADP-ribose polymers catalyzed by poly(ADP-ribose) polymerase-1 (PARP-1) regulates eucaryotic gene expression, including the NF-kappaB-dependent pathway. Here, we report the molecular mechanism by which PARP-1 activates the sequence-specific binding of NF-kappaB to its oligodeoxynucleotide. We co-incubated pure recombinant human PARP-1 and the p50 subunit of NF-kappaB (NF-kappaB-p50) in the presence or absence of beta NAD(+) in vitro. Electrophoretic mobility shift assays showed that, when PARP-1 was present, NF-kappaB-p50 DNA binding was dependent on the presence of beta NAD(+). DNA binding by NF-kappaB-p50 was not efficient in the absence of beta NAD(+). In fact, the binding was not efficient in the presence of 3-aminobenzamide (3-AB) either. Thus, we conclude that NF-kappaB-p50 DNA binding is protein-poly(ADP-ribosylation dependent. Co-immunoprecipitation and immunoblot analysis revealed that PARP-1 physically interacts with NF-kappaB-p50 with high specificity in the absence of beta NAD(+). Because NF-kappaB-p50 was not an efficient covalent target for poly(ADP-ribosylation, our results are consistent with the conclusion that the auto-poly(ADP-ribosyl)ation reaction catalyzed by PARP-1 facilitates the binding of NF-kappaB-p50 to its DNA by inhibiting the specific protein-protein interactions between NF-kappaB-p50 and PARP-1. We also report the activation of NF-kappaB(-)DNA binding by the automodification reaction of PARP-1 in cultured HeLa cells following exposure to H2O2. In these experiments, preincubation of HeLa cells with 3-AB, prior to oxidative damage, strongly inhibited NF-kappaB activation in vivo as well.