Regulation of the DNA-degrading enzyme TREX1 by intramembrane proteolysis and its implications for human auto-immune diseases as well as a model for ER proteostasis

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Mutations in the Three prime repair exonuclease 1 (TREX1) gene lead to several auto-inflammatory diseases including Aicardi-Goutières syndrome (AGS). TREX1 is a DNA-degrading enzyme, which can clear DNA from the cytosol. From a cell’s perspective, DNA in the cytosol can indicate intrusion of a pathogen. Therefore, DNA-sensing mechanisms like the cGAS-STING pathway have evolved triggering defence mechanisms like production of interferons. However, overactivation of these pathways by accumulating DNA, e.g. in the absence of TREX1, leads to immunopathology.TREX1 is synthesised as a tail-anchored protein which is attached to the ER membrane by a C-terminal transmembrane domain (TMD). This is in contradiction to several reports detecting TREX1 also in the nucleus. We have identified TREX1 as novel substrate of the ER-localised intramembrane protease Signal peptide peptidase (SPP). Cleavage by SPP releases a soluble form of TREX1 into the cytosol. We found that TREX1 can exist constitutively in the cleaved soluble as well as a membrane-bound form. However, TREX1 cleavage can also result in its rapid further degradation so that SPP also regulates total cellular TREX1 levels. These findings suggest a major regulatory impact of SPP on TREX1. As SPP mediates the transition between both forms, the analysis of SPP-deficient cells and mice provides a system where generation of soluble TREX1 is prevented. We plan to analyse where and under which conditions TREX1 cleavage takes place, how this influences the stability of the cleavage product and how this is connected to nuclear translocation. A central question is how the release of TREX1 from the membrane impacts on its capability to degrade cytosolic DNA resulting from different sources, including retrotransposons. Thinking this further, we will also study how absence or presence of cytosolic TREX1 modulates the initiation of interferon responses by exogenously introduced, but also pathogen-derived DNA or DNA released following irradiation like upon radiotherapy. This will also be analysed in a novel mouse model solely expressing soluble TREX1. Strong support that TREX1 proteolysis is a biologically relevant process is provided by an AGS-causing mutation in the TREX1 TMD, which significantly enhances cleavability of TREX1. We will investigate how the modulated processing of this and further TREX1 TMD mutations is connected to disease pathogenesis. With regard to the protease SPP, many aspects of substrate selection and further handling of cleavage products remain open to date. We consider TREX1 as a suitable model substrate to approach these SPP-related mechanistic questions to better understand SPP as part of ER proteostasis pathways. Altogether, this project will provide essential insights into cell biological mechanisms regulating nucleic acid-driven immune responses as well as how a dysregulation of these processes can lead to human disease.