Understanding the molecular pathways that underpin production, sensing and protection against aldehydes

短链醛(最重要的是甲醛)是制造中常规使用的高活性分子，在生物实验室中作为固定剂很常见。令人担忧的是，这些因素也是内源性的，包括作为代谢途径(如酒精代谢)和细胞分化事件的副产品。甲醛是DNA损伤的一个强有力的来源，大多数人认为它是DNA交联剂，会扭曲结构并阻止DNA的分离。这会导致突变，阻止我们DNA的准确读取和复制。幸运的是，细胞拥有一种“两层”保护机制，可以限制这些分子的破坏作用。第一层代表能有效解毒甲醛的代谢酶。第二种是由DNA损伤修复蛋白组成，在损伤发生后修复损伤。虽然这种冗余意味着具有单一突变的个体可以保持保护状态，但多种酶的突变可能导致：贫血、早衰(加速衰老)、骨髓衰竭、神经退化、癌症、发育异常、肝和肾衰竭以及严重体重下降。在缺乏双层保护的情况下，甲醛的整体生理后果已经得到了深入的研究，但我们仍然对甲醛破坏的具体细胞过程知之甚少，也不知道其来源。这个项目将研究细胞如何对甲醛做出反应以及甲醛是在哪里生产的这些基本问题。我们目前有两条证据可以用来解决这个问题。首先，存在对甲醛压力敏感的特定途径，这些途径向转录网络发送信号，并驱动基因表达的变化。其次，在不同的转录状态下，有局部甲醛产生的迹象。这个项目将对这两种现象进行扩展，以建立一幅细胞中甲醛的来源和反应的图景。我将利用先进的基因编辑和测序技术进行CRISPR基因敲除筛查，目的是识别对甲醛敏感的途径，以及潜在的新的一级保护酶。这一结果的下游验证将首先在体外系统中进行，然后进入体内小鼠模型的生成，以确定它们的生理重要性。其次，我将使用先进的显微镜和化学探针工具来定位细胞中甲醛分解代谢的特定位置。通过了解使细胞对甲醛敏感的分子途径和位点，我们可以更广泛地了解甲醛诱导致病的机制。扩大对驱动甲醛应激的已知遗传因素的这一知识，对于预防和及早发现相关疾病以及开辟精确医学的途径至关重要。

Short chain aldehydes (most importantly formaldehyde) are highly reactive molecules used routinely in manufacturing, and are commonplace as fixative agents in biological laboratories. Worryingly, these factors are also produced endogenously, including as by-products of metabolic pathways (e.g., alcohol metabolism) and cell differentiation events. Formaldehyde is a potent source of DNA damage, most understood as a DNA cross-linking agent, distorting the structure and preventing the separation of DNA. This leads to mutagenic breaks, prevents accurate reading and replication of our DNA. Fortunately, cells possess a "two-tiered" protection mechanism that limits the damaging effects of these molecules. The first tier represents metabolic enzymes that actively detoxify formaldehyde. The second is made up of DNA damage repair proteins that fix the damage after it has happened. While this redundancy means individuals with a single mutation can remain protected, mutations in multiple enzymes can result in: anaemia, progeria (accelerated ageing), bone marrow failure, neurodegeneration, cancer, developmental abnormalities, liver and kidney failure, and severe weight loss. Where the overall physiological consequences of formaldehyde in the absence of two-tier protection have been heavily studied, we still know very little about the specific cellular process formaldehyde disrupts, nor of its origins. This project will look into the fundamental questions of how does the cell respond to formaldehyde and where is it produced? There are two current lines of evidence we can use to access this problem. Firstly, there are specific pathways sensitive to formaldehyde stress that are signalled to the transcriptional network and drive changes in gene expression. Secondly, there are signs of localised formaldehyde production in different transcriptional states. This project will expand on both of these phenomena to build a picture of the sources of and responses to formaldehyde in cells. I will employ advanced gene editing and sequencing technologies to perform a CRISPR knock out screen, with the aim of identifying pathways sensitive to formaldehyde, and potentially new tier 1 protection enzymes. Downstream validation of the results of this will be performed first in in vitro systems, progressing into the generation of in vivo mouse models to determine their physiological importance. Secondly, I will use advanced microscopy and chemical probe tools to localise specific sites of formaldehyde catabolism in cells. By understanding the molecular pathways and sites that render cells sensitive to formaldehyde, we can more broadly understand the mechanisms that underline formaldehyde induced morbidities. Expanding this knowledge of known genetic factors that drive formaldehyde stress, is an essential part in prevention and early detection of associated diseases, as well as opening avenues for precision medicine.