Characterisation of the physiological functions and regulatory networks of Signal Peptide Peptidase in the murine testis

• Applicant: Dr. Torben Mentrup
• Subject Area: Cell Biology ; Biochemistry
• Term: since 2025
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 565022475

The aspartyl intramembrane protease signal peptide peptidase (SPP) specifically cleaves selected transmembrane domains in the hydrophobic environment of the endoplasmic reticulum (ER) membrane. Based on this, important functions of SPP in the control of ER-associated degradation of proteins, but also the maturation of viruses and general ER proteostasis were discovered. However, little is known about the physiological function and possible regulation of this enzyme in the living organism, as mice with systemic loss of SPP are not viable. In this project, we aim to analyse the functions of SPP in the murine testis in more detail. This organ is particularly suitable for analysis due to the selective expression of another member of the aspartyl intramembrane protease family, the SPP-like 2c (SPPL2c) protease, in this tissue. In addition to their common localisation in the ER, both proteases have partially overlapping substrate spectra, but differ strongly in terms of their regulatory mechanisms and their incorporation into high-molecular protein complexes. In the murine testis, SPP is found in acrosomal compartments from the spermatid stage onwards, but also in mature spermatozoa. To determine the function of SPP in male germ cells, we have developed a conditional knockout strategy in which the expression of SPP can be specifically abrogated in these cells. While the genetic depletion of SPPL2c only causes a mild reproductive phenotype in mice, the selective loss of SPP expression in male germ cells leads to an almost complete loss of epididymal spermatozoa. This is not caused by a general cytotoxic effect of SPP ablation, as there are no signs of increased ER stress or apoptosis in the testes of corresponding knockout animals. Our preliminary findings therefore point to a specific function of SPP in late spermatogenesis or spermiation. We aim to analyse this in more detail by the examination of established, but also novel SPP-dependent processes. To this end, the substrate repertoire of SPP in the murine testis will be determined using mass spectrometric methods. Identified substrates will then be mechanistically linked to the observed phenotypes. In addition, we aim to gain new insights into the regulation and non-proteolytic functions of this important enzyme by characterising its in vivo interactome. In both cases, identified substrates or SPP-interactors will also be examined with regard to their interaction with SPPL2c in order to further elaborate functional differences between the two proteases. From the chosen approach, we expect not only detailed results on the physiological function of SPP in relation to male gametogenesis, but also a better understanding of the molecular processes that control the proteolytic activity of this enzyme in other tissues.

• DFG Programme: Research Grants