Enzymatic and non-enzymatic functions of the SUMO isopeptidase USPL1 in vivo

• Applicant: Privatdozent Dr. Klaus-Peter Knobeloch
• Subject Area: Cell Biology ; General Genetics and Functional Genome Biology ; Biochemistry
• Term: from 2018 to 2022
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 398279577

Protein modification by ubiquitin (Ub) and ubiquitin like proteins (UBLs) affect pleiotropic biological functions and are counteracted by Ub/UBL proteases. SUMO modification is one of the best studied UBL-modifications. In eukaryotes SUMO deconjugation by SUMO/Sentrin proteases (SenP1-7) is best characterized and mouse k.o. studies showed the critical role of SenPs in physiological processes including hypoxia, hematopoiesis, embryogenesis and inflammation. It was initially thought that SUMO-deconjugation is exclusively mediated by SenPs. However, recently USPl1 was identified as a non-SenP SUMO-specific isopeptidase which also possesses non-enzymatic functions. Yet, the function of USPL1 is poorly explored, only a handful of publications are listed in Pubmed and the physiological relevance is entirely undefined. We have now generated Uspl1fl/fl conditional and Uspl1C224A knock-In mice expressing an inactive enzyme. Our unpublished preliminary work shows that (i) Lack of USPL1 causes early embryonic lethality and apoptosis in embryonic stem cells. (ii) Induced deletion in adult Uspl1fl/fl MxCre mice results in lethal anemia. When the hematopoietic defect was rescued by bone marrow transplantation, lethal hepatic steatosis was observed. (iii) Analysis of Uspl1fl/flCD4Cre mice shows a critical role of USPL1 in T cell development. In contrast, selective inactivation of only the enzymatic activity in Uspl1C224A mice did not affect viability or cause a dramatic phenotype.We will now follow up on the physiological analysis and get insight into the molecular mechanisms underlying enzymatic and non-enzymatic functions of USPL1 in vivo. Therefore we will1. Determine which cell types and pathways require USPL1 and characterize the physiological and molecular functions of USPL1 in vivo by (i) Evaluating The biological and molecular function of USPL1 in ES cells and embryogenesis. (ii) Analyzing physiological and molecular functions of USPL1 in the liver, terminally differentiated cells and different organs. (iii) Define critical molecular- and enzymatic functions of USPL1 within the hematopoietic compartment.2. Identify physiological and molecular functions and substrates of the deSUMOylation activity of USPL1 and determine potential compensatory mechanisms. To this end we will (i) perform an in depth pathological and histological analysis of Uspl1C224A mice and follow up on the observed shift in T/B cell composition. (ii) Analyse potential compensatory upregulation of other SUMO proteases and if applicable address redundancy by the generation of double k.o mice. To circumvent potential adaptive mechanisms we will combine the Uspl1fl/fl with the Uspl1C224A allele allowing timely controlled inactivation of only the protease activity of USPL1. We are confident to shed light on the physiological and molecular function of USPL1 and will equip the scientific community with novel mouse models and tools for the analysis of this weakly explored SUMO-protease.

• DFG Programme: Research Grants