In vivo substrate spectrum and functions of the intramembrane protease signal peptide peptidase (SPP)

• Applicant: Professor Dr. Bernd Schröder
• Subject Area: Biochemistry
• Term: since 2025
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 555305071

Signal peptide peptidase (SPP) is an ER-localised aspartyl intramembrane protease which was initially identified based on its ability to process select signal peptides within the ER membrane. Most known endogenous substrates of SPP are tail-anchored (TA) proteins which fulfil the general substrate requirements of SPP with a single-span transmembrane segment in type II orientation (cytosolic N-terminus) and a short luminal domain. It remains an open question if mammalian SPP can cleave multi-span membrane proteins which was reported for its yeast orthologue Ypf1. As constitutive SPP knockout mice are perinatally lethal, our current knowledge on SPP substrates and functions is exclusively derived from cell line-based systems, which indicates a role of this protease in ER-associated protein degradation (ERAD). To decipher the impact of SPP on cellular proteostasis in an in vivo context, we have created and validated an inducible murine knockout model using a tamoxifen-activated Cre-ERT2 transgene. Postnatal loss of SPP is well tolerated so that these mice can be used for phenotypic studies. We will comprehensively characterise these mice including a broad immunologic phenotyping reflecting that homologues of SPP, the SPP-like proteases, play important roles in development and function of different immune cells. Preliminary data suggest alterations of serum cholesterol homeostasis upon loss of SPP, which will be analysed further as it may be relevant under patho-physiological conditions. Therefore, SPP-deficient mice will be challenged with a cholesterol-enriched Western type diet. Connected to this, we will investigate how SPP deficiency modulates cholesterol handling pathways at the organismic as well at the cellular level. We observed that SPP deficiency in liver leads to major changes in the membrane proteome. A significant number of single-span transmembrane proteins was enriched which represent SPP candidate substrates based on their topology. In addition, several polytopic membrane proteins were accumulated in the knockout tissue. Since loss of SPP also led to ER stress and an unfolded protein response (UPR), we plan to systematically dissect primary proteomic changes due to loss of SPP-mediated proteolysis from secondary effects. Proteolysis of confirmed substrate candidates will be in-depth characterised in cell-based systems. This approach will provide a list of validated of SPP in vivo substrates and essential functional insights. Furthermore, it will help to clarify the capability of mammalian SPP to process polytopic membrane proteins. A key question will be to delineate the molecular connection between SPP deficiency and the induction of a UPR. Therefore, ER stress pathways will be systemically characterised in inducible SPP KO mice. In addition, primary cells and cell line-based models will be used to link ER stress and/or the phenotypes described above with specific substrates of SPP and its proteostatic function.

• DFG Programme: Research Grants