Structural and functional analysis of the related transcriptional enhancer factor-1 and NF-κB interaction.
Structural and functional analysis of the related transcriptional enhancer factor-1 and NF-κB interaction.
复制标题
相关转录增强因子-1 和 NF-κB 相互作用的结构和功能分析。
DOI：
10.1152/ajpheart.00069.2013
复制
发表时间：
2014
期刊：
影响因子：
--
通讯作者：
Guo,Shaodong
中科院分区：
文献类型：
--
作者：
Ma,Jieliang;Zhang,Li;Tipton,AaronR;Wu,Jiaping;Messmer-Blust,AngelaF;Philbrick,MelissaJ;Qi,Yajuan;Liu,Song-Tao;Liu,Hongsheng;Li,Jian;Guo,Shaodong
相关的转录增强因子-1促进缺氧诱导因子-1α(α)的基因转录，促进血管内皮细胞的新生。缺氧和炎性因子肿瘤坏死因子-α均可调节HIF-1α的基因表达，但两者如何协同调控HIF-1α基因转录尚不清楚。在这里，我们发现rTEF-1与核因子-κB的p65亚基相互作用，p65亚基是肿瘤坏死因子-α的主要介质。RTEF-1可增强HIF-1α启动子活性，而p65亚基的表达则抑制这一作用。相比之下，p65基因的敲除显著增强了RTEF-1对HIF-1α启动子活性的刺激(7倍)。通过细胞免疫共沉淀实验和谷胱甘肽转移酶(GST)下拉实验证实RTEF-1与p65之间存在物理相互作用。对RTEF-1晶体结构的计算分析表明，RTEF-1的保守表面可能通过位于T347、Y349、R351和Y352的四个氨基酸残基与P65相互作用。我们进行了定点突变和谷胱甘肽转移酶下拉试验，证明rTEF-1中的Tyr352(Y352)是rTEF-1和p65-NF-κB复合体形成的关键位点。RTEF-1基因Y352位丙氨酸突变破坏了RTEF-1与p65的相互作用。此外，RTEF-1的表达降低了肿瘤坏死因子-α诱导的HIF-1α启动子活性、IL-1β和IL-6mRNA水平，但当Y352突变为丙氨酸时，RTEF-1的作用基本消失。这些结果表明，RTEF-1通过Y352与p65-NF-κB相互作用，并相互拮抗HIF-1α的转录激活，提示了RTEF-1调控基因表达的新机制，将缺氧与炎症联系起来。
The related transcriptional enhancer factor-1 (RTEF-1) increases gene transcription of hypoxia-inducible factor 1α (HIF-1α) and enhances angiogenesis in endothelium. Both hypoxia and inflammatory factor TNF-α regulate gene expression of HIF-1α, but how RTEF-1 and TNF-α coordinately regulate HIF-1α gene transcription is unclear. Here, we found that RTEF-1 interacts with p65 subunit of NF-κB, a primary mediator of TNF-α. RTEF-1 increased HIF-1α promoter activity, whereas expression of p65 subunit inhibited the stimulatory effect. By contrast, knockdown of p65 markedly enhanced RTEF-1 stimulation on the HIF-1α promoter activity (7-fold). A physical interaction between RTEF-1 and p65 was confirmed by coimmunoprecipitation experiments in cells and glutathioneS-transferase (GST)-pull-down assays. A computational analysis of RTEF-1 crystal structures revealed that a conserved surface of RTEF-1 potentially interacts with p65 via four amino acid residues located at T347, Y349, R351, and Y352. We performed site-directed mutagenesis and GST-pull-down assays and demonstrated that Tyr352 (Y352) in RTEF-1 is a key site for the formation of RTEF-1 and p65-NF-κB complex. An alanine mutation at Y352 of RTEF-1 disrupted the interaction of RTEF-1 with p65. Moreover, expression of RTEF-1 decreased TNF-α-induced HIF-1α promoter activity, IL-1β, and IL-6 mRNA levels in cells; however, the effect of RTEF-1 was largely lost when Y352 was mutated to alanine. These results indicate that RTEF-1 interacts with p65-NF-κB through Y352 and that they antagonize each other for HIF-1α transcriptional activation, suggesting a novel mechanism by which RTEF-1 regulates gene expression, linking hypoxia to inflammation.