DNA damage induced phosphorylation and regulation of NF-kappaB

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特殊蛋白质群，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-kB）家族作为免疫、炎症和应激反应的调节剂具有特别重要的作用。由于NF-kB发挥的核心作用，它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损伤的影响与感染和炎症引起的反应的研究程度不同，但它非常重要，并且可以通过暴露于环境基因毒素发生。许多依靠DNA损伤作为杀伤肿瘤细胞机制的常见癌症疗法也激活了该蛋白家族。此外，炎症或衰老过程中活性氧（ROS）的产生也会导致DNA损伤并触发NF-kB的“激活”。NF-kB激活对这些刺激的反应可能会有很大的不同，这取决于刺激的类型、发生刺激的细胞类型以及同时被激活的其他蛋白质的存在。这些差异表现为NF-kB对不同基因的激活或抑制，这可以根据环境而变化。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、炎症蛋白的产生和ROS的产生的影响。DNA损伤也是如此，不同类型的DNA损伤和DNA损伤剂对NF-kB功能的影响非常不同；这反过来又不同于对炎症刺激反应激活的NF-kB。在很大程度上，这些差异可以通过添加小功能化学基团（如磷酸盐）来修饰蛋白质结构来解释，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编码形式。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ptm对该蛋白的水平和复杂性比之前认为的要大得多。在本提案中，我们将扩展这一分析，并使用最先进的技术来表征响应不同类型的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同一分子上还是分别出现在不同分子上（产生不同修饰蛋白质的混合物）。与Eyers小组的分析并行，Perkins小组将确定这些ptm的功能和重要性。这包括酶的鉴定，被称为激酶，它调节在NF-kB蛋白的不同位置添加磷酸基团。此外，根据Eyers实验室的分析，NF-kB蛋白RelA中的氨基酸将发生突变，使其不能再以这种方式进行修饰，从而可以评估这些修饰对NF-kB dna损伤诱导反应调节的重要性。这些实验将为炎症、衰老和癌症化疗期间DNA损伤对NF-kB的调节提供新的见解。它们还将为未来研究NF-kB活性的其他方面（不同的刺激、背景和激活途径）提供一个模板，同时也可以作为刺激反应网络的一个更广泛的相关例子。

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.