Role of ovarian tumor domain proteases in NF-kB regulation and cell survival in the Helicobacter pylori-infected gastric epithelium

幽门螺杆菌是世界上一半的人口中存在的微生物，是某些人发生胃病的危险因素。细菌定植于胃粘膜上皮并引发氧化应激，这可导致极化上皮的破坏和持续性炎症。H.幽门相关性炎症主要由转录因子核因子κ B（NF-kB）的激活和趋化因子的释放引发。此外，NF-kB调节促进胃癌细胞存活的抗凋亡基因的表达。蛋白质修饰，包括通过共价缀合的单链或多聚泛素链的可逆修饰，对于NF-κ B系统的控制特别重要。此外，NF-kB信号转导的减弱和终止受许多机制调节，包括去泛素化酶（DUB）的作用，特别是卵巢肿瘤结构域蛋白酶（OTU）家族，其从底物中去除泛素或干扰与多聚泛素链的结合。值得注意的是，由人类基因组编码的DUB在包括胃癌在内的多种癌症中过表达、突变或下调。有趣的是，H。幽门螺杆菌和一些其它革兰氏阴性菌通过脂多糖代谢物ADP-β-D-甘露庚糖、肿瘤坏死因子受体相关因子（TRAF）相互作用蛋白（TIFA）独特地激活。在这里，我们表征了分子机制，并发现TIFA在H. pylori诱导的经典和替代NF kB途径。关于通过蛋白质泛素化（去泛素化）调节NF-κ B信号传导、其对基因表达（例如抗凋亡基因）的影响以及确保在H.幽门感染在这个项目中，我们的目标是破译不同OTU对经典和替代NF-κ B调节和H.幽门感染我们的单层2D胃类器官与柱状上皮形态类似于人类上皮，并允许我们阐明OTU在原代细胞中的功能特异性。在这里，我们将使用CRISPR/Cas9技术为特定的OTU生成一组敲除细胞/类器官，同时还将在DUB特异性CRISPR/Cas9筛选中识别其他DUB。最后，我们将检查人胃活检组织中NF-κ B调节OTU的表达，以将改变的表达与疾病状态相关联。揭示DUB酶在H.幽门螺杆菌感染将是了解人类胃病发生的一个重大进展。

The microorganism Helicobacter pylori is present in half of the world's population and is a risk factor for the occurrence of gastric diseases for some people. The bacteria colonise the epithelium of the gastric mucosa and trigger oxidative stress, which can lead to destruction of the polarised epithelium and persistent inflammation. H. pylori-associated inflammation is mainly initiated by the activation of the transcription factor Nuclear factor kappa-B (NF-kB) and the release of chemokines. In addition, NF-kB regulates the expression of anti-apoptotic genes that promote the survival of gastric cancer cells. Protein modifications, including reversible modification by covalently conjugated mono- or polyubiquitin chains, are of particular importance for the control of the NF-kB system. In addition, the attenuation and termination of the NF-kB signal transduction is regulated by a number of mechanisms, including the action of deubiquitinylases (DUBs), particularly the ovarian tumour domain protease (OTU) family, which remove ubiquitin from substrates or interfere with binding to polyubiquitin chains. Remarkably, DUBs encoded by the human genome are overexpressed, mutated or downregulated in a variety of cancers, including gastric cancer. Interestingly, H. pylori and some other Gram negative bacteria activate uniquely via a lipopolysaccharide metabolite, ADP-β-D-manno-heptose, the tumour necrosis factor receptor-associated factor (TRAF)-interacting protein (TIFA). Here, we characterised the molecular mechanism and found that TIFA has a dual function in H. pylori-induced classical and alternative NF kB pathways. Little is known about the regulation of NF-kB signaling by (de)ubiquitinylation of proteins, its effects on gene expression (e.g. anti-apoptotic genes) and the complex network of negative feedback loops that ensure termination of the NF-kB response in H. pylori infection. In this project, we aim to decipher the extent of the suppressive/regulatory capacity of different OTUs on the molecular dynamics of classical and alternative NF-kB regulation and apoptotic cell death in H. pylori infection. Our single-layered 2D gastric organoids with a columnar epithelial morphology resemble human epithelium and allow us to elucidate the functional specificity of OTUs in primary cells. Here we will use CRISPR/Cas9 technology to generate a set of knock-out cells/organoids for specific OTUs, but also identify other DUBs in a DUB-specific CRISPR/Cas9 screen. Finally, we will examine the expression of NF-kB-regulating OTUs in human gastric biopsies to correlate altered expression with disease status. Unravelling the role of DUB enzymes in NF-kB control, cell survival and gastric pathology in H. pylori infection would be a significant advance in understanding the emergence of human gastric disease.