Design and in vivo assembly of switchable protein-protein interactions for transcription regulation

生物学的许多方面都依赖于分子聚集在一起对特定信号做出反应。这方面的一个重要例子是基因调控。我们身体中的绝大多数细胞含有相同的DNA，但它们会根据收到的信号启动特定的基因。当基因被不适当地开启或关闭时，细胞的行为可能会改变，有可能导致癌症或糖尿病等疾病。使我们能够人工开启和关闭基因的系统将是可以在广泛的医疗和生物技术应用中使用的强大工具。基因由一种名为转录因子(TF)的蛋白质调控，这种蛋白质结合到DNA上的特定位置，通过招募其他蛋白质来开启基因，或者通过阻止这些蛋白质的结合来关闭基因。其中很多都是对小分子有反应的开关。例如，一种名为Lac Repressor的细菌转运蛋白控制着一种参与分解糖的基因。当糖存在时，一个小分子与Lac抑制物结合，改变蛋白质的形状，启动基因。像Lac抑制子这样的蛋白质被用作控制工程细胞中基因表达的工具，例如已经被改变为生产人类蛋白质的细菌。然而，我们利用日益复杂的生物化学来设计细胞的能力和愿望意味着现在迫切需要新的可切换的转录因子。理想情况下，它们不会干扰正常的细胞生物学，即所谓的正交性；它们应该具有可预测和可调节的性质，导致一组可靠的生物部分可以适应不同的用途；它们应该由可以进入细胞的无毒小分子控制，因此可能被用作药物。我们已经制定了规则，允许在实验室设计和合成称为卷曲线圈的蛋白质。卷曲的线圈可以被设计成以不同的方式组装；例如，将2、3、4或更多的蛋白质聚集在一起，这些蛋白质可以紧密地或弱地结合在一起。我们已经证明，这些设计的盘绕线圈可以在细胞内用来聚集控制基因所需的蛋白质。通过改变卷曲线圈组件之间相互作用的强度，我们可以控制目标基因的开启或关闭程度。这些新的转录因子具有可预测和可调节的特性，但目前它们不能被小分子控制来充当开关。在这项拟议的工作中，我们的目标是在我们的发现的基础上，生产只有在小分子存在的情况下才能组装的卷曲线圈。为此，我们将(I)削弱通常将其结合在一起的相互作用，(Ii)为小分子药物创造一个结合的空间。在适当的条件下，小分子将紧密而具体地填补所产生的缺口，并将充当拼图中缺失的一部分，使卷曲的线圈形成并聚集基因调控所需的蛋白质。为了找到这样的条件，我们将测试一系列范围广泛的线圈，这些线圈具有不同形状、大小和化学成分的空心铁芯。我们还将测试具有潜在互补性的小分子的多样化样本。出于这个原因，这项工作是与阿斯利康合作完成的，阿斯利康拥有大量小分子文库，并拥有引导我们走向其中最有希望的小分子文库的专业知识。该项目的结果将是一系列紧凑的、众所周知的盘绕线圈，其细胞内外的组装可以通过添加小分子或药物来控制。这项工作还将发展未来制造新开关所需的知识和程序。我们将使用这些系统来生产能够在细菌和人类细胞中打开和关闭基因的转录因子，通过Medimmune，我们将把这些工具应用于目前生产具有医学意义的生物分子的问题。

Many aspects of biology rely on molecules coming together in response to specific signals. An important example of this is gene regulation. The vast majority of cells in our body contain the same DNA, but they turn on specific genes in response to the signals that they receive. When genes are switched on or off inappropriately the behaviour of cells can change, potentially giving rise to diseases such as cancer or diabetes. Systems that enable us to turn genes on and off artificially would be powerful tools that could be used in a wide range of medical and biotechnological applications.Genes are regulated by proteins called transcription factors (TFs), which bind to specific sites on DNA, and either turn genes on by recruiting other proteins, or turn genes off by blocking the binding of such proteins. Many of these TFs are switches that respond to small molecules. For example, a bacterial TF called Lac repressor controls a gene involved in breaking down sugar. When the sugar is present a small molecule binds to Lac repressor, changes the shape of the protein and turns the gene on. Proteins like the Lac repressor are used as tools to control gene expression in engineered cells, such as bacteria that have been altered to produce human proteins. However, our ability and desire to engineer cells with increasingly complicated biochemistry means that there is now a pressing need for new switchable TFs. Ideally, these would not interfere with normal cell biology, referred to as being orthogonal; they should have predictable and tuneable properties, leading to a reliable set of biological parts that can be adapted for different uses; and they should be controlled by small non-toxic molecules that can enter cells, and could therefore potentially be used as drugs.We have developed rules that allow proteins called coiled coils to be designed and synthesised in the lab. Coiled coils can be designed to assemble in different ways; e.g., to bring together 2, 3, 4 or more proteins, which can bind to each other tightly or weakly. We have shown that these designed coiled coils can be used inside cells to bring together the proteins needed to control a gene. By altering the strength of the interaction between the components of the coiled coil we can control how much the targeted gene is turned on or off. These new TFs have predictable and tuneable properties, but they cannot currently be controlled by small molecules to act as a switch. In the proposed work we aim to build on our findings to produce coiled coils that assemble only in the presence of a small molecule.To do this, we will "hollow out" the centre of a coiled coil (i) to weaken the interactions that normally hold it together, and (ii) to generate a space for small-molecule drugs to bind. Under the right conditions, the small molecule will tightly and specifically fill the gap created, and will act as the missing piece of the jigsaw to allow the coiled coil to form and bring together the proteins needed for gene regulation. To find such conditions, we will test a large and diverse range of coiled coils that have hollowed cores of different shapes, sizes and chemistries. We will also test a diverse sample of potentially complementary small molecules. For this reason, the work is being done in collaboration with AstraZeneca, who have large libraries of small molecules, and the expertise to guide us towards the most promising of these. The outcome of this project will be a series of compact, well-understood coiled coils whose assembly inside or outside of cells can be controlled by adding a small molecule or drug. The work will also develop the knowledge and procedures needed to make new switches in future. We will use these systems to produce TFs that can turn genes on and off in both bacteria and in human cells, and with Medimmune we will apply these tools to current problems in the production of biological molecules of medical interest.