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

用于转录调控的可切换蛋白质-蛋白质相互作用的设计和体内组装

• 批准号: BB/S002820/1
• 负责人: Nigel Savery
• 金额: $ 98.88万
• 依托单位: University of Bristol
• 依托单位国家: 英国
• 项目类别: Research Grant
• 财政年份: 2018
• 资助国家: 英国
• 起止时间: 2018 至 无数据
• 项目状态: 已结题
• 来源: https://gtr.ukri.org/projects?ref=BB%2FS002820%2F1
• 关键词: Design; vivo; assembly; switchable; protein

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.

生物学的许多方面都依赖于分子聚集在一起以响应特定的信号。一个重要的例子是基因调控。我们身体中的绝大多数细胞都含有相同的DNA，但它们会根据接收到的信号开启特定的基因。当基因被不恰当地打开或关闭时，细胞的行为就会发生变化，可能导致癌症或糖尿病等疾病。让我们能够人工开启和关闭基因的系统将是一种强大的工具，可以广泛应用于医学和生物技术领域。基因由一种称为转录因子（TF）的蛋白质调节，转录因子与DNA上的特定位点结合，通过招募其他蛋白质来开启基因，或者通过阻断这些蛋白质的结合来关闭基因。这些TF中的许多是响应小分子的开关。例如，一种被称为Lac阻遏物的细菌TF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阻遏物结合，改变蛋白质的形状并打开基因。Lac阻遏物等蛋白质被用作控制工程细胞中基因表达的工具，例如已经被改变以产生人类蛋白质的细菌。然而，我们的能力和愿望工程细胞越来越复杂的生物化学意味着现在迫切需要新的可切换TF。理想情况下，它们不会干扰正常的细胞生物学，称为正交;它们应该具有可预测和可调的特性，从而产生一组可靠的生物学部分，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，因此有可能被用作药物。我们已经制定了规则，允许在实验室中设计和合成称为卷曲螺旋的蛋白质。盘绕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或微弱地相互结合。我们已经证明，这些设计的卷曲螺旋可以在细胞内用于将控制基因所需的蛋白质聚集在一起。通过改变卷曲螺旋组件之间相互作用的强度，我们可以控制靶基因的开启或关闭程度。这些新的TF具有可预测和可调节的特性，但它们目前不能被小分子控制以充当开关。在这项工作中，我们的目标是建立在我们的研究结果的基础上，产生只有在小分子存在的情况下才能组装的卷曲螺旋。为此，我们将“挖空”卷曲螺旋的中心（i）以削弱通常将其保持在一起的相互作用，（ii）为小分子药物提供结合空间。在适当的条件下，小分子将紧密而特异地填充所产生的差距，并将作为拼图的缺失部分，允许卷曲螺旋形成并将基因调控所需的蛋白质聚集在一起。为了找到这样的条件，我们将测试大量不同的线圈，这些线圈具有不同形状，尺寸和化学性质的中空芯。我们还将测试各种可能互补的小分子样品。出于这个原因，这项工作是与阿斯利康合作完成的，阿斯利康拥有大量的小分子库，以及指导我们走向其中最有前途的专业知识。该项目的成果将是一系列紧凑的，易于理解的卷曲螺旋，其在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TF，这些TF可以在细菌和人类细胞中打开和关闭基因，并且使用Medimmune，我们将把这些工具应用于生产具有医学意义的生物分子的当前问题。

项目成果

Rational Design of Phosphorylation-Responsive Coiled Coil-Peptide Assemblies.

• DOI: 10.1021/acssynbio.3c00064
• 发表时间: 2023-04-21
• 期刊: ACS SYNTHETIC BIOLOGY
• 影响因子: 4.7
• 作者: Thompson, Harry F.;Beesley, Joseph L.;Langlands, Hannah D.;Edgell, Caitlin L.;Savery, Nigel J.;Woolfson, Derek N.
• 通讯作者: Woolfson, Derek N.

Design and Selection of Heterodimerizing Helical Hairpins for Synthetic Biology.

• DOI: 10.1021/acssynbio.3c00231
• 发表时间: 2023-06-16
• 期刊: ACS SYNTHETIC BIOLOGY
• 影响因子: 4.7
• 作者: Smith, Abigail J. J.;Naudin, Elise A. A.;Edgell, Caitlin L. L.;Baker, Emily G. G.;Mylemans, Bram;FitzPatrick, Laura;Herman, Andrew;Rice, Helen M. M.;Andrews, David M. M.;Tigue, Natalie;Woolfson, Derek N. N.;Savery, Nigel J. J.
• 通讯作者: Savery, Nigel J. J.