Investigating regulation and function of the cytosine deaminase APOBEC3A during cell cycle re-entry

我们的身体对抗病毒感染的一种方式是，当病毒进入我们的细胞并试图复制时，攻击病毒基因组(DNA或RNA，取决于病毒)。参与这种免疫反应的一种蛋白质APOBEC3A(A3A)修饰胞嘧啶，胞嘧啶是DNA和RNA的四个组成部分之一，导致病毒基因的错误(突变)和断裂。A3A通常不会在我们的细胞中发现高水平，除非在某些被称为巨噬细胞的特殊免疫细胞中。它是在感染时被激活的，在保护我们免受一系列病毒感染方面发挥着重要作用，但它也在包括湿疹和牛皮癣在内的炎症条件下被激活。尽管A3A有助于保护我们免受病毒感染，但这种保护是有代价的，因为我们和其他人已经证明，它可以逆转我们自己的基因，产生导致癌症的突变。许多研究证实，这一过程发生在很大比例的癌症中，特别是那些发生在口腔和喉咙、肺、乳腺、膀胱癌和子宫颈癌的组织(上皮细胞)。A3A不仅在癌症发展过程中突变了我们的DNA，而且似乎在患者接受化疗时，A3A可以继续以这种无赖的方式发挥作用；导致耐药性，并最终导致治疗失败。这一知识激发了学术界和工业界开发A3A抑制剂的倡议；A3A抑制剂是一种可以在接受化疗的患者中阻止这种突变活动的药物，从而防止肿瘤对这种疗法产生耐药性。虽然这种方法有可能改善数百万癌症患者的预后，但关于A3A是如何控制的，以及它在正常、健康的上皮细胞中发挥的功能，我们还不清楚，A3A突变的癌症就是从这些上皮细胞发展而来的。如果没有这些知识，我们几乎不知道是什么触发了A3A的异常活动，或者抑制A3A活动可能会对患者产生什么副作用。在这项提案中，我们根据我们在培养的人类上皮细胞中研究A3A所获得的三个关键发现，提出了一系列实验来解决这些问题。1)我们发现，通过在上皮细胞中模拟伤口愈合反应，我们可以将A3A基因的水平切换到比以前在这些细胞中看到的水平高得多，并且随着这些细胞复制它们的DNA，这一水平保持在非常高的水平，此时DNA可能容易受到A3A恶意活动的影响。2)通过删除上皮细胞中的A3A基因，我们发现了A3A在调节这些细胞分裂速度方面的一个以前未知的角色，如果我们要预测靶向A3A的癌症治疗的效果，这个角色是至关重要的。3)最近的研究表明，A3A可以修饰许多细胞信使RNA，这些信使RNA是允许我们的基因翻译成蛋白质的中间转录产物，但这一活动的意义尚不清楚。我们观察到当我们激活上皮细胞中的A3A时，这种RNA编辑活动被强烈地诱导。基于这些新的观察，我们将使用我们独特的工具来详细地确定A3A基因是如何在被刺激分裂的上皮细胞中被开启的，并将确定A3A在调节这一过程中所起的作用。我们将对A3A介导的RNA编辑事件进行全面调查，并将检验我们的假设，即这一活动允许A3A改变关键蛋白质的合成速度。这个项目将导致我们对A3A调节和功能的认识发生阶段性变化，它在正常上皮生物学以及从炎症到病毒感染和癌症的病理学中的重要性。如果我们要成功地利用A3A作为药物靶点，这些知识将是至关重要的。它还将解决一个基本问题，即A3A介导的RNA编辑在控制我们的基因如何表达方面所起的作用。

One way in which our bodies fight viral infections is to attack viral genomes (either DNA or RNA, depending on the virus) when viruses enter our cells and attempt to replicate. One protein involved in this immune response, APOBEC3A (A3A) modifies cytosine, one of the four building blocks of DNA and RNA, causing errors (mutations) and breaks in viral genes. A3A is not normally found at high levels in our cells except in certain specialised immune cells called macrophages. It is switched on in response to infection and plays an important role in protecting us from a range of viruses, however it is also activated in inflammatory conditions including eczema and psoriasis. Although A3A helps to defend us from viral infections, this protection comes at a cost, as we and others have shown that it can turn against our own genes, generating mutations that cause cancer. Numerous studies have confirmed that this process occurs in a large proportion of cancers, particularly those arising in the tissues (epithelia) that line the mouth and throat, lung, breast, bladder and cervix. Not only does A3A mutate our DNA during cancer development but it appears that A3A can continue to act in this rogue fashion while patients are receiving chemotherapy; driving drug-resistance and ultimately, treatment failure. This knowledge has stimulated initiatives in academia and industry to develop A3A inhibitors; drugs that could block this mutagenic activity in patients receiving chemotherapy, thereby preventing tumours from becoming resistant to the therapy. While this approach holds the potential to improve outcomes for millions of cancer patients, there is much we do not yet know about the way in which A3A is controlled and about the functions that it performs in the normal, healthy epithelial cells from which A3A-mutated cancers develop. Without this knowledge, we have little idea of what triggers rogue A3A activity, or what the side-effects of inhibiting A3A activity might be in patients. In this proposal, we set out a series of experiments to address these questions, based on three key findings that we have made from studying A3A in cultured human epithelial cells. 1) We have discovered that by mimicking a wound-healing response in epithelial cells, we can switch the A3A gene on to levels far higher than those previously seen in these cells and that remains at very high levels as these cells replicate their DNA, a time at which the DNA is potentially vulnerable to rogue A3A activity. 2) By deleting the A3A gene in epithelial cells, we have uncovered a previously unknown role for A3A in regulating the rate at which these cells divide, a role that is critical to understand if we are to anticipate the effects of targeting A3A for cancer therapy. 3) Recent studies have demonstrated that A3A can modify many cellular messenger RNAs, the intermediate transcripts that allow our genes to be translated into proteins but the significance of this activity remains unclear. We observe a very strong induction of this RNA-editing activity when we activate A3A in epithelial cells.Based on these novel observations, we will use our unique tools to identify in detail how the A3A gene is switched on in epithelial cells that have been stimulated to divide and will establish the role that A3A plays in regulating this process. We will conduct a comprehensive survey of A3A-mediated RNA editing events and will test our hypothesis that this activity allows A3A to change the rate at which key proteins are made.This project will result in a step-change in our knowledge of A3A regulation and function, its importance in normal epithelial biology and in pathologies ranging from inflammation to viral infections and cancer. This knowledge will be vital if we are to successfully harness A3A as a drug target. It will also address a fundamental question regarding the role of A3A-mediated RNA editing in controlling how our genes are expressed.