Unravelling SWI/SNF ARID1A/B paralogs function at sequence resolution

在序列分辨率下揭示 SWI/SNF ARID1A/B 旁系同源物功能

• 批准号: BB/Y004477/1
• 负责人: Jyoti Choudhary
• 金额: $ 113.67万
• 依托单位: Institute of Cancer Research
• 依托单位国家: 英国
• 项目类别: Research Grant
• 财政年份: 2024
• 资助国家: 英国
• 起止时间: 2024 至 无数据
• 项目状态: 未结题
• 来源: https://gtr.ukri.org/projects?ref=BB%2FY004477%2F1
• 关键词: Unravelling; SWI; SNF; ARID1A; paralogs

Paralog proteins emerged through gene duplication events, they are very similar in sequence and structure and have related functions, but small and subtle differences in their sequences can lead to specific modulation of their roles. More than 60% of human proteins have paralogs and they are prevalent in chromatin protein complexes. Systematic approaches for sequence to function mapping are required to disentangle the specific biological roles and behaviours of the paralog pairs, and lack of scalable methods for this has limited our understanding of the molecular basis of diverging paralog functions. The overarching aim of this project is to understand differential sequence-function relationships of paralog protein pair ARID1A/1B. These proteins are subunits of DNA-binding multiprotein assemblies called SWI/SNF complexes that are important for many essential functions, including cell proliferation, cell cycle control, response to DNA damage and organism development. Most proteins work by associating with other proteins, so characterising how proteins interact is important for fully understanding how they perform their roles. In this project we will identify the proteins that ARID1A and 1B interact with, mapping the domains or motifs that mediate the interactions, investigating the effect of mutations in their sequence on cell growth and finally, integrating all the data to construct models that can be helpful for understanding the mechanisms of ARID1A and 1B function.To identify ARID1A and 1B associated proteins we will purify ARID1A/B in conditions that maintain the native interactions with antibodies that specifically recognise them and use a technique called mass spectrometry to identify proteins that co-purify with them. We will also use mass spectrometry to identify post-translational modifications, small chemical "flags" that can regulated different aspects of protein function, like protein activity, interactions or locatlisation amongst others. To identify binding domains we will use peptides, or short protein fragments, covering the entire length of ARID1A and 1B arrayed on a paper membrane. A cell extract is added, and proteins that can interact with the peptides remain bound to the membrane. Each peptide spot will then be analysed by mass spectrometry to identify the bound proteins. This strategy is optimal to detect binding dependent on short motifs. To map binding domains that depend on the 3D structure of ARID1A/B we will fix the interactions inside the cell using a "molecular glue" that binds proteins that are very close to each other. We will use mass spectrometry to identify the regions of the proteins that were linked together. To identify aminoacids in ARID1A/B that are important for cell growth when the paralog is absent, we will mutate each aa sequentially to alanine and monitor cell proliferation. Finally, we will consolidate all the data to produce a graph that represents an integrated view of the knowledge acquired. This will be useful to generate hypothesis on how ARID1A/B differentially perform their specific roles.

旁系同源蛋白是通过基因复制事件出现的，它们在序列和结构上非常相似，并且具有相关的功能，但它们序列上微小而微妙的差异可以导致其作用的特定调节。超过 60% 的人类蛋白质具有旁系同源物，并且它们普遍存在于染色质蛋白质复合物中。需要用于序列到功能映射的系统方法来解开旁系同源对的特定生物学作用和行为，并且缺乏可扩展的方法限制了我们对不同旁系同源功能的分子基础的理解。该项目的首要目标是了解旁系同源蛋白对 ARID1A/1B 的差异序列功能关系。这些蛋白质是 DNA 结合多蛋白组装体的亚基，称为 SWI/SNF 复合物，对许多基本功能很重要，包括细胞增殖、细胞周期控制、对 DNA 损伤的反应和生物体发育。大多数蛋白质通过与其他蛋白质结合来发挥作用，因此表征蛋白质如何相互作用对于充分了解它们如何发挥作用非常重要。在这个项目中，我们将识别 ARID1A 和 1B 相互作用的蛋白质，绘制介导相互作用的结构域或基序，研究其序列突变对细胞生长的影响，最后整合所有数据来构建有用的模型为了了解 ARID1A 和 1B 功能的机制。为了鉴定 ARID1A 和 1B 相关蛋白，我们将在保持与特异性识别它们的抗体天然相互作用的条件下纯化 ARID1A/B，并使用称为质谱的技术识别与其共纯化的蛋白质。我们还将使用质谱法来识别翻译后修饰，即可以调节蛋白质功能不同方面的小化学“标志”，例如蛋白质活性、相互作用或定位等。为了识别结合域，我们将使用肽或短蛋白质片段，覆盖排列在纸膜上的 ARID1A 和 1B 的整个长度。添加细胞提取物，并且可以与肽相互作用的蛋白质仍然结合在膜上。然后通过质谱分析每个肽点以鉴定结合的蛋白质。该策略最适合检测依赖于短基序的结合。为了绘制依赖于 ARID1A/B 3D 结构的结合域，我们将使用一种“分子胶”来修复细胞内的相互作用，这种“分子胶”可以结合彼此非常接近的蛋白质。我们将使用质谱来识别连接在一起的蛋白质区域。为了鉴定 ARID1A/B 中在旁系同源物缺失时对细胞生长重要的氨基酸，我们将按顺序将每个氨基酸突变为丙氨酸并监测细胞增殖。最后，我们将整合所有数据以生成一个图表，该图表代表所获取知识的综合视图。这对于生成 ARID1A/B 如何差异化执行其特定角色的假设很有用。