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

• Applicant: Professor Dr. Michael Naumann
• Subject Area: Gastroenterology
• Term: since 2023
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 518060957

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.

• DFG Programme: Research Grants