Regulation and cleavage specificity of the ER-resident intramembrane proteases SPPL2c and SPP

• Applicant: Dr. Torben Mentrup
• Subject Area: Biochemistry ; Cell Biology
• Term: since 2019
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 431664610

The Signal Peptide Peptidase (SPP)/SPP-like (SPPL) family is comprised of five aspartic intramembrane proteases that fulfil diverse (patho)physiological functions ranging from regulation of immune cell homeostasis to the control of atherosclerosis. In contrast to the closely related presenilins, SPP/SPPL proteases exclusively process transmembrane segments with type II topology. Since the discovery of SPP/SPPL intramembrane proteases in the beginning of this century, SPPL2c has remained the least characterised family member. Due to its intron-less gene structure, it was even believed to represent a non-expressed pseudogene. In contrast to this, we could recently demonstrate endogenous expression of SPPL2c in the endoplasmic reticulum (ER) of male germ cells where it is able to process selected tail-anchored (TA) proteins. Interestingly, substrate spectra of SPPL2c and the other ER-resident family member SPP only partially overlapped suggesting a yet not understood specificity of both proteases. In vivo, SPPL2c is exclusively expressed in the ER of elongated spermatids in the murine and human testis where it mediates the turnover of the TA proteins Syntaxin-8 and phospholamban thereby modulating organelle remodelling and sperm motility. By this means, SPPL2c contributes to the regulation of male fertility. Even though our initial analysis of SPPL2c provided deep insights into its biological functions, it also raised significant questions. How are substrates discriminated from non-substrates by SPPL2c and SPP? How can we explain differential processing of TA proteins by these two very similar proteases? And, most importantly, how is intramembrane protease activity controlled to maintain a stable pool of TA proteins? To answer these questions, we will perform extensive mutagenetic studies on transmembrane domains of selected TA proteins to elucidate factors deciding over their general cleavability by SPP and SPPL2c. However, our major focus will be set on the regulation on the protease side. So far, SPP/SPPL proteases are believed to be constitutively active enzymes without any known regulators. In contrast to this, in preliminary experiments we identified the so far undescribed C11orf94 protein as a novel SPPL2c interacting protein. This interaction has major effects on both partners: While co-expression of SPPL2c stabilises C11orf94, presence of the latter almost completely blocks processing of all tested TA proteins by SPPL2c but not by SPP. Based on our strong data from cell-based experiments, we aim to analyse the relevance of the SPPL2c-C11orf94-axis in vivo in the murine testis using knockout mouse models for both proteins. In addition, we will perform IP-MS experiments to characterise the further interactome of SPPL2c in the murine testis to further expand our knowledge about the protein environment governing the reactive milieu of this protease and to gain valuable insights into regulatory mechanisms of intramembrane proteolysis in general.

• DFG Programme: Research Grants