当前位置: X-MOL 学术J. Am. Soc. Mass Spectrom. › 论文详情
Our official English website, www.x-mol.net, welcomes your feedback! (Note: you will need to create a separate account there.)
DNA Binding and Phosphorylation Regulate the Core Structure of the NF-κB p50 Transcription Factor.
Journal of the American Society for Mass Spectrometry ( IF 3.1 ) Pub Date : 2018-06-05 , DOI: 10.1007/s13361-018-1984-0
Matthias Vonderach 1 , Dominic P Byrne 2 , Perdita E Barran 3 , Patrick A Eyers 2 , Claire E Eyers 1
Affiliation  

The NF-κB transcription factors are known to be extensively phosphorylated, with dynamic site-specific modification regulating their ability to dimerize and interact with DNA. p50, the proteolytic product of p105 (NF-κB1), forms homodimers that bind DNA but lack intrinsic transactivation function, functioning as repressors of transcription from κB promoters. Here, we examine the roles of specific phosphorylation events catalysed by either protein kinase A (PKAc) or Chk1, in regulating the functions of p50 homodimers. LC-MS/MS analysis of proteolysed p50 following in vitro phosphorylation allows us to define Ser328 and Ser337 as PKAc- and Chk1-mediated modifications, and pinpoint an additional four Chk1 phosphosites: Ser65, Thr152, Ser242 and Ser248. Native mass spectrometry (MS) reveals Chk1- and PKAc-regulated disruption of p50 homodimer formation through Ser337. Additionally, we characterise the Chk1-mediated phosphosite, Ser242, as a regulator of DNA binding, with a S242D p50 phosphomimetic exhibiting a > 10-fold reduction in DNA binding affinity. Conformational dynamics of phosphomimetic p50 variants, including S242D, are further explored using ion-mobility MS (IM-MS). Finally, comparative theoretical modelling with experimentally observed p50 conformers, in the absence and presence of DNA, reveals that the p50 homodimer undergoes conformational contraction during electrospray ionisation that is stabilised by complex formation with κB DNA. Graphical Abstract ᅟ.

中文翻译:

DNA结合和磷酸化调节NF-κBp50转录因子的核心结构。

已知NF-κB转录因子被广泛地磷酸化,其动态位点特异性修饰调节其使二聚体与DNA相互作用的能力。p50是p105(NF-κB1)的蛋白水解产物,形成与DNA结合但缺乏固有反式激活功能的同型二聚体,可作为κB启动子转录的阻遏物。在这里,我们检查了由蛋白激酶A(PKAc)或Chk1催化的特定磷酸化事件在调节p50同型二聚体功能中的作用。体外磷酸化后,蛋白水解的p50的LC-MS / MS分析使我们可以将Ser328和Ser337定义为PKAc和Chk1介导的修饰,并指出另外四个Chk1磷酸位点:Ser65,Thr152,Ser242和Ser248。天然质谱(MS)揭示了Chk1和PKAc调控的通过Ser337破坏p50同二聚体的形成。此外,我们将Chk1介导的磷酸化位点Ser242表征为DNA结合的调节剂,而S242D p50磷酸模拟物表现出DNA结合亲和力降低> 10倍。使用离子迁移率质谱(IM-MS)进一步探索了包括S242D在内的拟磷酸化p50变体的构象动力学。最后,在不存在和存在DNA的情况下,使用实验观察到的p50构象体进行的比较理论建模表明,p50同二聚体在电喷雾电离过程中会经历构象收缩,这种收缩可通过与κBDNA形成复合物来稳定。图形摘要ᅟ。S242D p50磷酸酯类似物的DNA结合亲和力降低> 10倍。使用离子迁移率质谱(IM-MS)进一步探索了包括S242D在内的拟磷酸化p50变体的构象动力学。最后,在不存在和存在DNA的情况下,使用实验观察到的p50构象体进行的比较理论建模表明,p50同二聚体在电喷雾电离过程中会经历构象收缩,这种收缩可通过与κBDNA形成复合物来稳定。图形摘要ᅟ。S242D p50磷酸酯类似物的DNA结合亲和力降低> 10倍。使用离子迁移率质谱(IM-MS)进一步探索了包括S242D在内的拟磷酸化p50变体的构象动力学。最后,在不存在和存在DNA的情况下,使用实验观察到的p50构象体进行的比较理论建模表明,p50同二聚体在电喷雾电离过程中会经历构象收缩,这种收缩可通过与κBDNA形成复合物来稳定。图形摘要ᅟ。揭示p50同二聚体在电喷雾电离过程中经历构象收缩,并通过与κBDNA形成复合物而稳定。图形摘要ᅟ。揭示p50同二聚体在电喷雾电离过程中经历构象收缩,并通过与κBDNA形成复合物而稳定。图形摘要ᅟ。
更新日期:2018-06-05
down
wechat
bug