Substrate recognition and binding by Signal Peptide Peptidase-like 2 (SPPL2) family

• Applicant: Professorin Dr. Regina Fluhrer
• Subject Area: Biochemistry
• Term: from 2015 to 2023
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 263531414

Signal peptide peptidase (SPP) and the homologous SPP-like (SPPL) proteases, SPPL2a, SPPL2b, SPPL2c and SPPL3, belong to the family of GxGD proteases and thus represent one prototype of intramembrane cleaving enzymes. In the first funding period we applied a proteome wide screen and identified a variety of new candidate substrates for SPPL2a, SPPL2b and, in addition, also for SPPL2c, a so far orphan protease. Moreover, we identified three new SPPL2a/b substrates by candidate approach and determined the C-terminal SPPL2 cleavage sites in these substrates. Based on the analysis of chimeric constructs from the established SPPL2 substrate TNFalpha and the non-substrate Bri3 as well as of TNF mutant substrates we postulate a multistep cleavage model for substrate processing by SPPL2b, in which every step demands individual determinants. While binding of full length substrates to the enzyme seems to occur rather non-specifically, substrate recognition in the active centre is favoured by a short luminal substrate domain. The initial cleavages at the C-terminal end of the substrates TM domain are determined by amino acids 8-14 of the substrates luminal juxtamembrane domain. Whether the substrate subsequently undergoes consecutive cleavage within its TM domain is determined by the helical stability of the TM domain. And finally, the stability of the cleavage products and thus most likely also their putative signaling capacity depends on an S-palmitoylation at the cytosolic TM boundary of the substrate.Goal 1: In the new funding period we will validate the newly identified SPPL2 candidate substrates in our established cell culture models as well as in our newly established SPPL2a and SPPL2b knock out cells. Goal 2: Since our model of SPPL2 cleavage is mainly based on the analysis of TNFalpha and superficial knowledge on Bri2, we will apply targeted changes to other substrates to confirm the generality of our results. Finally, we aim to convert the non-substrate Bri3 by applying a minimal set of mutations into a bonafide SPPL2 substrate.Goal 3: Having elucidated the substrate intrinsic determinants required for efficient processing by SPPL2 proteases, we will now investigate the specific prerequisites for substrate recognition within the enzyme. While SPPL2b preferentially process substrates with a short luminal juxtamembrane domain, SPPL3 accepts substrates independent of their ectodomain length. Based on this, we will establish SPPL2/SPPL3 chimeric proteins to identify the domains within SPPL2 that are responsible for selection of substrates with short ectodomains. Altogether, project P2 will provide a more detailed insight into the substrate determinates that allow recognition and cleavage by SPPL2 proteases and will help to understand how protease and substrate interact. Thus, project P2 contributes to the two major aims of the research group and helps to further unravel the molecular mechanism of intramembrane cleavage.

• DFG Programme: Research Units
• Subproject of: FOR 2290: Understanding Intramembrane Proteolysis