Investigating the targets and biological roles of the deubiquitylase USP43

研究去泛素化酶 USP43 的靶标和生物学作用

• 批准号: BB/S017062/1
• 负责人: Simon Cook
• 金额: $ 44.37万
• 依托单位: Babraham Institute
• 依托单位国家: 英国
• 项目类别: Research Grant
• 财政年份: 2019
• 资助国家: 英国
• 起止时间: 2019 至 无数据
• 项目状态: 已结题
• 来源: https://gtr.ukri.org/projects?ref=BB%2FS017062%2F1
• 关键词: Investigating; targets; biological; roles; deubiquitylase

The cells in our body must be able to sense and respond to changes in their environment (hormones, UV damage, toxins) to maintain the healthy function of tissues and organs. Failure to do so progressively undermines cellular 'fitness' contributing to age-related declines in cell and tissue function that drive the normal ageing process and can contribute to age-related diseases such as chronic inflammation, cancer and dementia.Cells respond to tissue damage or infection by increasing the abundance of key proteins and enzymes that mitigate that damage. They do this through the activation of intracellular signalling pathways which transmit information into the cell. When a cell receives this signal the genes that code for enzymes are 'read' by 'transcription factors', discrete proteins that bind to DNA and transcribe the DNA information into RNA molecules, which are in turn 'translated' into the relevant proteins and enzymes. One such transcription factor, called 'NFkappaB' (NFkB), coordinates cellular responses to an inflammatory signal called 'TNF'. When cells are exposed to TNF, NFkB becomes activated as a result of phosphorylation - the attachment of phosphate groups; this requires two enzymes called the IkB kinase or IKKs. We have deleted the genes for both IKKs in human colon cells using CRISPR gene editing. By analyzing the changes in abundance of RNA molecules we found that TNF-NFkB signalling activates the gene that codes for a protein called USP43. TNF cannot increase USP43 abundance in cells that lack IKKs. We also noted that cells that lack IKK have increased cell-to-cell contacts (i.e. closer physical contact with neighbouring cells) and increased abundance of a protein called E-cadherin that mediates cell:cell adhesion signals. We therefore examined how cells respond to increasing cell density, which promotes cell:cell contacts. This led us to discover that USP43 abundance also increases with increasing cell density and that USP43 is found in cells close to E-cadherin at sites of cell-cell contacts. Formation of correct cell:cell contacts is vital for the formation and maintenance of complex tissues. If it fails it can cause inflammation, including inflammatory bowel disease (IBD). Indeed, genetic defects in components of the TNF-IKK-NFkB pathway and E-cadherin have both been shown to contribute to IBD syndromes so it is very striking that USP43 abundance is controlled by both pathways and is found at sites of cell:cell contact in colon cells. Indeed, recent data from another lab has found mutations in the gene for USP43 which impair USP43 activity in patients with inflammation. For these reasons we are interested in the function of the USP43 protein, which belongs to a family of enzymes that cleave a protein called ubiquitin from other proteins. Many proteins in our cells are 'tagged' by the addition of ubiquitin and this changes the properties of such proteins, regulating their activity, directing them for destruction or directing them to specific compartments within the cell. USP43 reverses the addition of ubiquitin to proteins. We suspect that USP43 controls the activity or abundance of proteins that are critical for TNF-IKK-NFkB inflammatory signalling and E-cadherin signalling. In this project we will define how USP43 abundance is controlled, identify the targets of USP43 (those proteins that USP43 removes ubiquitin from) and other USP43 interacting proteins that may control its functions. We will delete the USP43 gene from human cells so that we can assess the role of the USP43 protein in regulating inflammatory signalling, cell:cell contacts, cell survival and cell division. The results will shed new light on how inflammation and tissue structure are controlled and may tell us whether USP43 is a possible new drug target for inflammatory disease.

我们体内的细胞必须能够感知环境的变化（激素、紫外线损伤、毒素）并做出反应，以维持组织和器官的健康功能。如果不这样做，就会逐渐破坏细胞的“健康”，导致与年龄相关的细胞和组织功能下降，从而驱动正常的衰老过程，并可能导致慢性炎症、癌症和痴呆等与年龄相关的疾病。细胞通过增加减轻损伤的关键蛋白质和酶的丰度来对组织损伤或感染做出反应。它们通过激活将信息传递到细胞内的细胞内信号传导途径来做到这一点。当细胞接收到该信号时，编码酶的基因会被“转录因子”“读取”，“转录因子”是与 DNA 结合并将 DNA 信息转录为 RNA 分子的离散蛋白质，而 RNA 分子又被“翻译”为相关的蛋白质和酶。其中一种转录因子称为“NFkappaB”(NFkB)，可协调细胞对称为“TNF”的炎症信号的反应。当细胞暴露于 TNF 时，NFkB 由于磷酸化（磷酸基团的附着）而被激活；这需要两种称为 IkB 激酶或 IKK 的酶。我们使用 CRISPR 基因编辑技术删除了人类结肠细胞中两种 IKK 的基因。通过分析 RNA 分子丰度的变化，我们发现 TNF-NFkB 信号传导激活编码 USP43 蛋白质的基因。 TNF 不能增加缺乏 IKK 的细胞中 USP43 的丰度。我们还注意到，缺乏 IKK 的细胞细胞间接触增加（即与邻近细胞更紧密的物理接触），并且介导细胞间粘附信号的称为 E-钙粘蛋白的蛋白质丰度增加。因此，我们研究了细胞如何响应细胞密度的增加，从而促进细胞与细胞的接触。这使我们发现 USP43 丰度也随着细胞密度的增加而增加，并且 USP43 存在于细胞与细胞接触部位靠近 E-钙粘蛋白的细胞中。 正确的细胞与细胞接触的形成对于复杂组织的形成和维持至关重要。如果失败，可能会引起炎症，包括炎症性肠病（IBD）。事实上，TNF-IKK-NFkB 途径和 E-钙粘蛋白成分的遗传缺陷均已被证明会导致 IBD 综合征，因此非常引人注目的是，USP43 丰度受这两种途径控制，并且发现于结肠细胞中细胞与细胞接触的部位。事实上，另一个实验室的最新数据发现 USP43 基因突变会损害炎症患者的 USP43 活性。由于这些原因，我们对 USP43 蛋白的功能感兴趣，该蛋白属于将泛素蛋白从其他蛋白中裂解出来的酶家族。我们细胞中的许多蛋白质通过添加泛素而被“标记”，这改变了这些蛋白质的特性，调节它们的活性，引导它们被破坏或引导它们进入细胞内的特定区室。 USP43 逆转蛋白质中泛素的添加。我们怀疑 USP43 控制对 TNF-IKK-NFkB 炎症信号传导和 E-钙粘蛋白信号传导至关重要的蛋白质的活性或丰度。在这个项目中，我们将定义如何控制 USP43 丰度，确定 USP43 的靶标（USP43 从中去除泛素的蛋白质）和其他可能控制其功能的 USP43 相互作用蛋白。我们将从人类细胞中删除 USP43 基因，以便评估 USP43 蛋白在调节炎症信号、细胞与细胞接触、细胞存活和细胞分裂中的作用。这些结果将为炎症和组织结构的控制提供新的线索，并可能告诉我们 USP43 是否是炎症性疾病的可能新药物靶点。