DNA damage induced phosphorylation and regulation of NF-kappaB

DNA 损伤诱导 NF-kappaB 磷酸化和调节

• 批准号: BB/L009501/1
• 负责人: Claire Eyers
• 金额: $ 49.97万
• 依托单位: University of Liverpool
• 依托单位国家: 英国
• 项目类别: Research Grant
• 财政年份: 2014
• 资助国家: 英国
• 起止时间: 2014 至 无数据
• 项目状态: 已结题
• 来源: https://gtr.ukri.org/projects?ref=BB%2FL009501%2F1
• 关键词: DNA; damage; induced; phosphorylation; regulation

To respond to threats from the environment or infectious agents, organisms 'activate' specialized groups of proteins that regulate the response of different genes, thus allowing cells of the body to adapt and survive, or fight the infection. Among these types of proteins, the Nuclear Factor kappa B (NF-kB) family is of particular importance as a regulator of the immune, inflammatory and stress responses. Because of the central role it plays, NF-kB can respond to a large number of different stimuli that include bacteria, viruses, inflammatory proteins and cell stresses such as DNA damage. Although the effect of DNA damage is not studied to the same extent as the response due to infection and inflammation, it is of great importance and can occur through exposure to environmental genotoxins. Many common cancer therapies that rely on DNA damage as their mechanism of killing tumour cells also active this protein family. Moreover, the production of reactive oxygen species (ROS) during inflammation or as a result of ageing can also lead to DNA damage and trigger 'activation' of NF-kB. The consequences of NF-kB activation in response to these stimuli can vary enormously, depending on the type of stimulation, the cell type in which it is occurring and the presence of other proteins activated at the same time. These differences manifest themselves as activation or repression of different genes by NF-kB, which can vary according to the context. Thus the effect of activating NF-kB on the cell and the organism will also vary depending on the type of stimulus. These differences include effects on cell proliferation, survival, production of inflammatory proteins and generation of ROS. This is also true of DNA damage, where different types of DNA damage and DNA damaging agent can result in very different effects on NF-kB function; this in turn differs from NF-kB activated in response to inflammatory stimuli. To a large extent, these differences can be explained by modifications to the protein structure through addition of small functional chemical groups, such as phosphate, a process termed post-translational modification (PTM). Such PTM is a form of code that rapidly regulates protein function in cells. Preliminary data from the Eyers group, looking at the effect of the cytokine TNF on the NF-kB protein RelA, has indicated that the level and complexity of PTMs on this protein is much greater than previous thought. In this proposal we will extend this analysis and use state of the art techniques to characterise the multiple PTMs that occur in response to different types of DNA damage. This analysis will include not only the identification of the sites of modification but also how they change during time and whether they occur simultaneously on the same molecules or are present separately on different molecules (generating a mix of differently modified proteins). In parallel with the analysis in the Eyers group, the Perkins group will determine the function and importance of these PTMs. This includes the identification of the enzymes, called kinases that regulate the addition of phosphate groups to different positions in the NF-kB protein. Furthermore, based the analysis in the Eyers lab, amino acids in the NF-kB protein RelA will be mutated such that it can no longer be modified in this manner, thus allowing the importance of these modifications on the regulation of the DNA-damage-induced response of NF-kB to be assessed. These experiments will give new insights into the regulation of NF-kB by DNA damage during inflammation, ageing and cancer chemotherapy. They will also provide a template for future analysis to investigate other aspects of NF-kB activity (different stimuli, context and activation pathways) while also serving more generally as a relevant example of a stimulus responsive network.

为了应对来自环境或感染因子的威胁，生物体“激活”调节不同基因反应的专门蛋白质组，从而使身体细胞适应和生存，或对抗感染。在这些类型的蛋白质中，核因子κ B（NF-κ B）家族作为免疫、炎症和应激反应的调节剂是特别重要的。由于它所起的核心作用，NF-kB可以对大量不同的刺激做出反应，包括细菌，病毒，炎症蛋白和细胞应激，如DNA损伤。虽然DNA损伤的影响没有被研究到与感染和炎症反应相同的程度，但它非常重要，并且可以通过暴露于环境遗传毒素而发生。许多依赖DNA损伤作为其杀死肿瘤细胞机制的常见癌症疗法也激活了这种蛋白质家族。此外，在炎症过程中或由于衰老而产生的活性氧（ROS）也会导致DNA损伤并触发NF-κ B的“激活”。响应于这些刺激的NF-kB激活的结果可以变化很大，这取决于刺激的类型、发生的细胞类型以及同时激活的其他蛋白质的存在。这些差异表现为NF-κ B对不同基因的激活或抑制，其可以根据上下文而变化。因此，激活NF-kB对细胞和生物体的影响也将根据刺激的类型而变化。这些差异包括对细胞增殖、存活、炎性蛋白产生和ROS产生的影响。DNA损伤也是如此，其中不同类型的DNA损伤和DNA损伤剂可导致对NF-kB功能的非常不同的影响;这又不同于响应于炎症刺激而激活的NF-kB。在很大程度上，这些差异可以通过添加小的功能化学基团（如磷酸盐）对蛋白质结构进行修饰来解释，这一过程称为翻译后修饰（PTM）。这种PTM是一种快速调节细胞中蛋白质功能的代码形式。来自Eyers小组的初步数据，观察了细胞因子TNF对NF-kB蛋白RelA的影响，表明该蛋白上PTM的水平和复杂性比以前认为的要大得多。在本提案中，我们将扩展这种分析，并使用最先进的技术来检测响应于不同类型的DNA损伤而发生的多种PTM。这种分析不仅包括鉴定修饰位点，还包括它们如何随时间变化，以及它们是同时发生在相同分子上还是分别存在于不同分子上（产生不同修饰蛋白质的混合物）。与Eyers小组的分析同时，Perkins小组将确定这些PTM的功能和重要性。这包括鉴定被称为激酶的酶，这些酶调节磷酸基团添加到NF-κ B蛋白的不同位置。此外，基于Eyers实验室的分析，NF-kB蛋白RelA中的氨基酸将被突变，使得它不再能够以这种方式被修饰，从而允许评估这些修饰对调节DNA损伤诱导的NF-kB应答的重要性。这些实验将为炎症、衰老和癌症化疗期间DNA损伤对NF-κ B的调节提供新的见解。他们还将为未来的分析提供一个模板，以研究NF-kB活性的其他方面（不同的刺激，背景和激活途径），同时也更普遍地作为刺激响应网络的相关示例。

项目成果

DOSCATs: Double standards for protein quantification.

• DOI: 10.1038/srep45570
• 发表时间: 2017-04-03
• 期刊: Scientific reports
• 影响因子: 4.6
• 作者: Bennett RJ;Simpson DM;Holman SW;Ryan S;Brownridge P;Eyers CE;Colyer J;Beynon RJ
• 通讯作者: Beynon RJ

Temporal modulation of the NF-?B RelA network in response to different types of DNA damage

NF-κB RelA 网络响应不同类型 DNA 损伤的时间调节

• DOI: 10.1101/2020.08.11.246504
• 发表时间: 2020
• 作者: Campbell A

Use of the Polo-like kinase 4 (PLK4) inhibitor centrinone to investigate intracellular signaling networks using SILAC-based phosphoproteomics

使用 Polo 样激酶 4 (PLK4) 抑制剂 centrinone 通过基于 SILAC 的磷酸蛋白质组学研究细胞内信号网络

• DOI: 10.1101/2020.05.22.110767
• 发表时间: 2020
• 作者: Byrne D

Human CDK18 promotes replication stress signaling and genome stability.

• DOI: 10.1093/nar/gkw615
• 发表时间: 2016-10-14
• 期刊: Nucleic acids research
• 影响因子: 14.9
• 作者: Barone G;Staples CJ;Ganesh A;Patterson KW;Bryne DP;Myers KN;Patil AA;Eyers CE;Maslen S;Skehel JM;Eyers PA;Collis SJ
• 通讯作者: Collis SJ

cAMP-dependent protein kinase (PKA) complexes probed by complementary differential scanning fluorimetry and ion mobility-mass spectrometry.

• DOI: 10.1042/bcj20160648
• 发表时间: 2016-10-01
• 期刊: The Biochemical journal
• 作者: Byrne DP;Vonderach M;Ferries S;Brownridge PJ;Eyers CE;Eyers PA
• 通讯作者: Eyers PA