University thesis:
Dissertation, Universität Freiburg, 2022
Footnote:
Description:
Abstract: The following dissertation summarizes the biological functions of DPP9, the role of DPP9 in the DNA Damage Response, especially in regulating it's substrate, BRCA2 and contains a brief summary on one of the two Proteases of the SARS-CoV-2 Virus, namely PLpro.<br><br>In the work, focussed on the role of DPP9 in the DNA damage response, we identify the tumour suppressor BRCA2 as a novel DPP9 substrate. We propose that, like in the case of Syk, DPP9 requires FLNA to act as a recruitment platform and interacts with BRCA2 upon DNA damage. We show in crystal processing of BRCA2 N-terminal peptides, with the N-terminal dipeptidecaptured in the active site. We report that DPP9 depletion causes hypersensitivity towards genotoxic compounds and impaired HR. We demonstrate that DPP9 activity targets BRCA2 to the N-degron and that the removal of the N-terminal dipeptide is required for the formation of RAD51 foci, the downstream effect of BRCA2. To summarise, we propose a regulatory role for DPP9 in regulating the intracellular BRCA2 levels and shaping the BRCA2 interactome.<br><br>In the work focussed on the characterizion of the proteases of the SARS-CoV-2 virus, we display our results on a library of compounds against Mpro and PLpro of SARS-CoV-2, to identify compounds with anti-viral properties that could be utilized as therapeutics. Here, we characterised the purified enzymes and screened for lead-inhibitors. While no inhibitors were identified for PLpro, seven were identified for Mpro. Out of these seven, two showed promising results in lungs cells, however, the promising results were diminished in cells that were expressing the transmembrane protease, serine 2 (TMPRSS2), suggesting multiple entry pathways. This work also aimed to point out the challenges in the screening methods used in the early days of SARS-CoV-2 research. Without optimised animal models, the focus has been on cellular systems, with a controversy regarding their ability to simulate the infection. Moreover, based on the limitations of the systems used, artificial biases might be introduced that affect the conclusions of drug screens. We argue for testing the compounds with promising anti-viral properties first against cross-reactivity against host protease pathways, prior to clinical trials, to decrease negative outcomes of identified compounds due to false confidence based on cellular efficacy data