Investigating the targets and biological roles of the deubiquitylase USP43

我们体内的细胞必须能够感知环境（激素、紫外线损伤、毒素）的变化并作出反应，以维持组织和器官的健康功能。如果做不到这一点，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，而细胞和组织功能会推动正常的衰老过程，并可能导致与年龄相关的疾病，如慢性炎症、癌症和痴呆症。细胞对组织损伤或感染的反应是增加减轻损伤的关键蛋白质和酶的丰度。它们通过激活将信息传递到细胞内的细胞内信号通路来做到这一点。当细胞接收到这种信号时，编码酶的基因就会被“转录因子”“读取”，转录因子是一种与DNA结合的分离蛋白质，将DNA信息转录成RNA分子，RNA分子又被“翻译”成相关的蛋白质和酶。其中一种转录因子称为NFkappaB (NFkB)，它协调细胞对炎症信号TNF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；这需要两种酶，称为IkB激酶或IKKs。我们使用CRISPR基因编辑技术删除了人类结肠细胞中这两个IKKs的基因。通过分析RNA分子丰度的变化，我们发现TNF-NFkB信号激活了编码USP43蛋白质的基因。TNF不能在缺乏IKKs的细胞中增加USP43的丰度。我们还注意到，缺乏IKK的细胞增加了细胞间的接触（即与邻近细胞更紧密的物理接触），并增加了介导细胞粘附信号的e -钙粘蛋白的丰度。因此，我们研究了细胞如何对增加的细胞密度做出反应，这促进了细胞间的接触。这使我们发现USP43的丰度也随着细胞密度的增加而增加，并且USP43在细胞-细胞接触部位靠近E-cadherin的细胞中发现。正确细胞的形成：细胞接触对复杂组织的形成和维持至关重要。如果它失败了，它会引起炎症，包括炎症性肠病（IBD）。事实上，TNF-IKK-NFkB通路和E-cadherin的遗传缺陷都被证明有助于IBD综合征，因此非常惊人的是，USP43丰度是由这两种通路控制的，并且在结肠细胞的细胞接触部位被发现。事实上，最近来自另一个实验室的数据发现了USP43基因的突变，这种突变会损害炎症患者的USP43活性。由于这些原因，我们对USP43蛋白的功能很感兴趣，它属于一个酶家族，可以从其他蛋白质中切割一种叫做泛素的蛋白质。我们细胞中的许多蛋白质都被添加了泛素“标记”，这改变了这些蛋白质的性质，调节它们的活性，引导它们破坏或引导它们进入细胞内的特定隔间。USP43逆转了泛素在蛋白质中的添加。我们怀疑USP43控制着对TNF-IKK-NFkB炎症信号和E-cadherin信号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USP43丰度是如何控制的，确定USP43的靶标（USP43去除泛素的那些蛋白质）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，这样我们就可以评估USP43蛋白在调节炎症信号、细胞接触、细胞存活和细胞分裂中的作用。这些结果将揭示炎症和组织结构是如何被控制的，并可能告诉我们USP43是否可能是炎症疾病的新药物靶点。

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.