Bilateral BBSRC-SFI: Deciphering the function of the human Dihydrofolate reductase 2 gene

叶酸是B族维生素的一种，由一组小分子组成，人体几乎每个细胞都需要这些小分子来完成许多基本功能，包括合成细胞分裂所需的DNA。像所有的维生素一样，我们需要确保摄入足量的叶酸来维持我们的健康。叶酸不足与出生缺陷、先天代谢缺陷、神经问题、自闭症、脂肪肝疾病、与年龄有关的认知障碍以及许多癌症有关。我们需要了解使用叶酸的途径，即叶酸代谢，以及它是如何在出生前后的不同组织和发育阶段受到调节的。叶酸代谢的各个步骤是由一组特殊的蛋白质介导的，这些蛋白质被称为酶，其中最重要的是二氢叶酸还原酶（DHFR）。缺乏DHFR功能会抑制叶酸代谢并阻止细胞分裂——这在正常组织中是非常有害的，但可以作为一些治疗癌症药物的作用基础。DHFR也是进入叶酸代谢的途径，叶酸包含在强化食品和维生素补充剂中，用于预防脊柱裂等出生缺陷。尽管有公共卫生信息，许多妇女仍然在叶酸不足的情况下怀孕。对叶酸和人类健康的进一步研究对于在整个欧洲推行强制性强化政策的公共卫生讨论至关重要。值得注意的是，我们发现人类和其他灵长类动物在进化过程中获得了第二个DHFR基因DHFR2，而其他哺乳动物只有一个。对DHFR2的功能知之甚少，但我们发现该基因在许多组织中都有活性，遗传学研究表明DHFR2的改变可能与神经管缺陷（NTDs）的一组严重出生缺陷的风险增加有关，神经管缺陷是指大脑和脊髓发育的早期事件。这些发现表明DHFR2可能在发育过程中起关键作用。为了更好地了解人类叶酸代谢，重要的是研究DHFR2的作用，并询问它是否与DHFR具有相似或不同的功能。我们将研究DHFR2蛋白的丰度和位置如何随着干细胞分化并成为更专门的细胞类型（如神经元）而变化。我们将使用遗传工具从培养细胞中去除DHFR2，并研究其对细胞特性（如增殖和分化）以及叶酸代谢活性的影响。同时，我们将询问在不同条件下DHFR和DHFR2的相对水平和位置如何不同。这些知识将有助于理解它的作用。下一步将是研究DHFR2在发育过程中的作用，我们将发现DHFR2在人类胚胎中不同阶段的何处表达。为了将这项工作转移到活胚胎中，我们将产生新的小鼠品系，其中小鼠DHFR基因被人类DHFR或DHFR2或两者替换。这些小鼠模型将允许我们对每种蛋白质的功能提出详细的问题。人DHFR或DHFR2能代替小鼠蛋白吗？只有DHFR2的存在是否足以正常发育，还是这些胚胎在生长、神经发育和/或叶酸代谢方面表现出变化？通过这种方式，对只表达DHFR或DHFR2的“人源化”小鼠的研究将告诉我们这两种酶的个体功能，这在同时表达这两种酶的人类细胞中是很难解决的。在人类细胞和小鼠模型中确定了DHFR2的功能后，项目的最后一部分将更详细地研究该蛋白的调节。我们将测试它是否以不同的结构形式存在，它是否在特定的位点被修改，以及它的产生是如何被调节的。总的来说，这个项目将为人类健康至关重要的基本代谢途径提供新的见解。

Folates are a type of B vitamin that consists of a group of small molecules that are needed in nearly every cell in the body to fulfil a number of essential functions including synthesis of DNA for cell division. Like all vitamins, we need to ensure that we consume a sufficient amount of folate to maintain our health. Inadequate folate is implicated in birth defects, inborn errors of metabolism, neurological problems, autism, fatty liver disease, age-related and cognitive impairment and many cancers. We need to understand the pathway that uses folate, known as folate metabolism, and how it is regulated in different tissues and stages of development before and after birth.The individual steps of folate metabolism are mediated by a specialised set of proteins, called enzymes, one of the most important being Dihydrofolate Reductase (DHFR). Lack of DHFR function suppresses folate metabolism and prevents cells dividing - this is very harmful in normal tissue but can be exploited as the basis of action of some drugs for the treatment of cancer. DHFR is also the route of entry into folate metabolism of folic acid, which is included in fortified foods and vitamin supplements for prevention of birth defects such as spina bifida. Despite public health messages, many women still become pregnant with inadequate folate status. Further research on folate and human health is essential to inform the public health discussion on the introduction of mandatory fortification policies across Europe.Notably we found that humans and other primates have acquired a second DHFR gene, DHFR2, during evolution whereas other mammals have only one. Little is known about the function of DHFR2 but we find that the gene is active in many tissues and genetic studies show that alteration of DHFR2 may be linked to increased risk of a group of severe birth defects termed neural tube defects (NTDs), in which the early events of brain and spinal cord development fail. These findings suggest that DHFR2 may play a key role during development. To gain a better understanding of human folate metabolism it is important to investigate the role of DHFR2 and to ask whether it has similar or distinct functions to DHFR. We will study how DHFR2 protein abundance and location changes as stem cells differentiate and become more specialised cell types such as neurons. We will use genetic tools to remove DHFR2 from cells in culture and investigate the consequences for cellular properties such as proliferation and differentiation, as well as activity of folate metabolism. In parallel, we will ask how the relative levels and location of DHFR and DHFR2 differ in varying conditions. This knowledge will all help to understand what it does. The next step will be to investigate the role of DHFR2 during development and we will find out where DHFR2 is expressed in human embryos at differing stages. In order to move this work to living embryos we will generate new mouse strains in which the mouse DHFR gene is replaced with human DHFR or DHFR2 or both. These mouse models will allow us to ask detailed questions about the function of each protein. Can human DHFR or DHFR2 substitute for the mouse protein? Is the presence of only DHFR2 sufficient for normal development or do these embryos show changes in growth, neural development and/or folate metabolism? In this way, the study of 'humanised' mice that express only DHFR or DHFR2 will tell us about the individual functions of the enzymes that is difficult to address in human cells that have both.Having established the functions of DHFR2 in human cells and mouse models, the final part of the project will examine the regulation of the protein in more detail. We will test whether it is present in different structural forms, whether it is modified at particular sites and how its production is regulated. Overall, this project will give new insight into a fundamental metabolic pathway that is crucial for human health.