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; 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并响应其环境变化（激素，紫外线损伤，毒素），以维持组织和器官的健康功能。未能逐步破坏细胞“适应性”，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，并可能导致与年龄相关的疾病，例如慢性炎症，癌症和痴呆。细胞会对组织损伤的丰富性而造成损害，从而对组织损害或感染产生损害的损害。他们通过激活细胞内信号通路的激活来实现此信息，这些通路将信息传输到细胞中。当一个细胞接收到该酶代码的基因时，通过“转录因子”“读取”了与DNA结合并将DNA信息结合并将DNA信息转录为RNA分子的基因，这些蛋白依次将其“翻译”成相关的蛋白质和酶。一种称为“ NFKappab”（NFKB）的转录因子协调了对称为“ TNF”的炎症信号的细胞反应。当细胞暴露于TNF时，NFKB由于磷酸化而被激活 - 磷酸基团的附着；这需要两个称为IKB激酶或IKK的酶。我们使用CRISPR基因编辑删除了人类结肠细胞中两个IKK的基因。通过分析RNA分子丰度的变化，我们发现TNF-NFKB信号传导激活编码称为USP43的蛋白质的基因。 TNF不能增加缺乏IKK的细胞中的USP43丰度。我们还注意到，缺乏IKK的细胞具有增加细胞对细胞接触（即与邻近细胞的物理接触更紧密），并且增加了一种称为e-钙粘蛋白的蛋白质，该蛋白质介导细胞：细胞粘附信号。因此，我们研究了细胞如何响应增加的细胞密度，从而促进细胞：细胞接触。这使我们发现USP43丰度也随着细胞密度的增加而增加，并且在细胞细胞接触位点接近E-钙粘着蛋白的细胞中发现了USP43。 正确的细胞的形成：细胞接触对于复杂组织的形成和维持至关重要。如果失败会引起炎症，包括炎症性肠病（IBD）。实际上，TNF-IKK-NFKB途径和E-钙粘着蛋白的成分中的遗传缺陷均已证明有助于IBD综合征，因此USP43的丰度受两种途径控制，并且在结肠细胞中的细胞接触位点发现了USP43的丰度。实际上，另一个实验室的最新数据发现USP43基因中的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，该蛋白属于一个酶家族，该酶从其他蛋白质中裂解一种称为泛素的蛋白质。我们细胞中的许多蛋白质通过添加泛素“标记”，这会改变此类蛋白质的特性，调节其活性，将其引导去破坏或将其引导到细胞内的特定隔室。 USP43逆转将泛素添加到蛋白质中。我们怀疑USP43控制着对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。在此项目中，我们将定义如何控制USP43的丰度，确定USP43的靶标（USP43从中消除泛素的蛋白质）和其他USP43相互作用的蛋白质可能控制其功能。我们将从人细胞中删除USP43基因，以便我们可以评估USP43蛋白在调节炎症信号传导中的作用，细胞：细胞接触，细胞存活和细胞分裂。结果将为炎症和组织结构的控制方式提供新的启示，并可能告诉我们USP43是否是炎症性疾病的新药物靶标。