IDENTIFICATION OF NUCLEAR PATHWAYS THAT ARE CAUSALLY INVOLVED IN NUCLEOLAR TARGETING OF NF-KAPPAB/RELA.

结直肠癌是英国非吸烟人群中最常见的癌症死亡原因，也是英国人口的一个主要公共卫生问题。明确的证据表明，阿司匹林和相关药物可以预防结直肠癌，并使这种疾病消退。然而，阿司匹林类药物的潜力受到其毒性的限制。在这个实验室里，我们一直在进行研究，以了解阿司匹林样药物如何作用于结肠癌细胞，以便开发更有效和更具体的替代品。我们已经证明，这些药物会导致一种参与细胞生长和死亡调节的分子RelA，从细胞的细胞质室转移到核质室，然后再转移到一个叫做核仁的核室。我们还发现RelA从核质向核仁的移动对于阿司匹林样药物杀死结肠癌细胞的能力很重要。此外，我们已经证明，将RelA人工定位到核仁可以介导这种细胞类型的死亡。本研究的总体目标是了解阿司匹林如何导致RelA从核质转移到核仁。确定所涉及的细胞途径将允许设计预防/治疗药物，迫使RelA进入核仁室杀死结肠癌细胞。我们已经证明了一种叫做COMMD1的分子在导致RelA对阿司匹林产生反应时进入核核中很重要，COMMD1通过将一种叫做泛素的调节分子连接到RelA上起作用。本建议的具体目标是：1。确定与COMMD1一起在阿司匹林治疗后连接泛素与RelA的重要分子。我们将研究候选分子的作用，这些分子先前已被确定在泛素与RelA的连接中起作用。我们还将从阿司匹林处理过的细胞中分离COMMD1，并使用一种称为质谱法的技术来识别与COMMD1结合的分子。我们将研究阿司匹林如何影响这些分子，以及阿司匹林如何上调COMMD1.2。确定RelA中与泛素相关的特定部分对阿司匹林的反应以及这种联系的性质。泛素通常与特定位置的赖氨酸蛋白相连。我们将确定RelA的赖氨酸，泛素与阿司匹林反应有关。泛素也在赖氨酸分子上与自身连接形成链。我们将确定泛素上的哪一种赖氨酸对RelA的核仁易位至关重要，在其他方法中，使用可以可视化泛素化的单细胞试验。将泛素连接的RelA转运到核仁的蛋白质鉴定。当泛素与一个分子连接时，特定的蛋白质会与该分子结合。因此，我们认为当泛素与RelA结合时，特定分子结合RelA并将蛋白质转运到核仁。我们将使用标记方法和质谱法来鉴定特异性结合阿司匹林诱导的泛素连接RelA的蛋白质。这些互补的方法将使我们了解RelA如何在核质中被调节的基础科学，如何将泛素与RelA连接使其进入核仁以及类似RelA的蛋白质如何定位到细胞中的不同区室。更重要的是，这些研究可能揭示了一种化学方法来操纵这些途径来模拟阿司匹林对结肠癌细胞的影响。

Colorectal cancer is the most common cause of cancer death amongst the non-smoking population in the UK and is a major public health issue for the British population. Unequivocal evidence indicates that aspirin and related agents can prevent colorectal cancer and cause regression of this disease. However, the potential of aspirin-like agents is limited by their toxicity. In this lab, we have been undertaking studies to understand how aspirin-like agents act against colon cancer cells, in order to develop more effective and specific alternatives. We have shown that these agents cause a molecule involved in the regulation of cell growth and death, RelA, to move from the cytoplasmic compartment of the cell to the nucleoplasmic compartment then to a nuclear compartment called the nucleolus. We have also shown that movement of RelA from the nucleoplasm to the nucleolus is important for the ability of aspirin-like agents to kill colon cancer cells. Furthermore, we have demonstrated that artificially localising RelA to the nucleolus mediates the death of this cell typeThe overall objective of this study is to understand how aspirin causes RelA to move from the nucleoplasm to the nucleolus. Identifying the cellular pathways involved will allow the design of preventative/therapeutic agents that force RelA into the nucleolar compartment to kill colon cancer cells. We have already shown that a molecule called COMMD1 is important in causing RelA to go to the nucleolus in response to aspirin and that COMMD1 acts by linking a regulatory molecule called ubiquitin, to RelA.The specific objectives of this proposal are:1. To identify molecules that, alongside COMMD1, are important for linking ubiquitin to RelA after aspirin treatment. We will examine the role of candidate molecules that have previously been identified as playing a role in linking ubiquitin to RelA. We will also isolate COMMD1 from aspirin treated cells and use a technique called mass spectrometry to identify molecules that bind to COMMD1 in response to the agent. We will examine how aspirin effects these molecules and how aspirin upregulates COMMD1.2. Identification of the specific parts of RelA that are linked to ubiquitin in response to aspirin and the nature of this linkage. Ubiquitin is generally linked onto proteins at specific sites called lysines. We will identify the lysines of RelA that ubiquitin is linked to in response to aspirin. Ubiquitin also links to itself on lysine molecules to form chains. We will determine which lysine on ubiquitin is critical for nucleolar translocation of RelA using, amongst other approaches, a single cell assay where ubiquitination can be visualised.3. Identification of proteins that transport ubiquitin-linked RelA to the nucleolus. When ubiquitin is linked to a molecule, specific proteins bind to that molecule. Therefore, we suggest that when ubiquitin is linked to RelA, specific molecules bind RelA and transport the protein to the nucleolus. We will use a labelling approach and mass spectrometry to identify proteins that bind specifically to aspirin-induced, ubiquitin-linked RelA. These complementary approaches will allow us to understand the basic science of how RelA is regulated in the nucleoplasm, how linking ubiquitin to RelA causes it to go to the nucleolus and how proteins similar to RelA locate to different compartments in the cell. More importantly, these studies may reveal a way to chemically manipulate these pathways to mimic effects of aspirin on colon cancer cells.