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

幽门螺杆菌存在于世界上一半的人口中，是一些人患胃病的危险因素。这种细菌定植在胃粘膜的上皮细胞上，并引发氧化应激，这可能导致极化的上皮细胞被破坏和持续的炎症。幽门螺杆菌相关性炎症主要由转录因子核因子-kB的激活和趋化因子的释放引起。此外，核因子-kB还调节促进胃癌细胞存活的抗凋亡基因的表达。蛋白质修饰，包括共价共轭的单泛素链或多泛素链的可逆修饰，对于控制核因子-kB系统特别重要。此外，核因子-kB信号转导的减弱和终止受到许多机制的调节，包括去泛素酶(DUBS)的作用，特别是卵巢肿瘤结构域蛋白酶(OTU)家族，它们从底物中去除泛素或干扰与多泛素链的结合。值得注意的是，由人类基因组编码的Dub在包括胃癌在内的各种癌症中过度表达、突变或下调。有趣的是，幽门螺杆菌和其他一些革兰氏阴性细菌通过脂多糖代谢产物-β-D-甘露七糖-肿瘤坏死因子受体相关因子相互作用蛋白(TIFA)独特地激活。在此，我们描述了TIFA的分子机制，发现TIFA在幽门螺杆菌诱导的经典和替代的NFkB途径中具有双重功能。幽门螺杆菌感染过程中，通过蛋白质的(去)泛素化对核因子-kB信号的调控，对基因表达的影响(如抗凋亡基因)，以及确保终止核因子-kB反应的复杂的负反馈环网络，人们知之甚少。在这个项目中，我们的目标是破译不同的OTUS对幽门螺杆菌感染中经典和替代的核因子-kB调控以及细胞凋亡的分子动力学的抑制/调节能力的程度。我们的具有柱状上皮形态的单层2D胃有机体类似于人类上皮，并使我们能够在原代细胞中阐明OTUS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/有机物，但也会在特定于配音的CRISPR/Cas9屏幕中识别其他DUB。最后，我们将检测调节核因子-kB的OTUS在人类胃活检组织中的表达，以将表达变化与疾病状态相关联。揭示幽门螺杆菌感染中DuB酶在核因子-kB调控、细胞存活和胃病理中的作用，将是理解人类胃病发生的重要进展。

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.