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PD Dr. Bernd Schröder

Bernd Schröder
Bernd Schröder
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Intramembrane Proteases and Novel Lysosomal Membrane Proteins
Physiological functions of the intramembrane proteases SPPL2a and SPPL2b
The concept of regulated intramembrane proteolysis (RIP) has emerged over the last decades as a novel concept of cellular signalling. One group among the proteases being capable of cleaving substrates within the phospholipid bilayer are the signal-peptide-peptidase (SPP) and its homologues, the signal-peptide-peptidase-like proteins (SPPL2a, -2b, -2c, -3). Whereas SPPL2a is present in membranes of lysosomes/late endosomes, SPPL2b was reported to reside at the plasma membrane as well as in endosomal compartments. To date, only TNFα, Fas Ligand (FasL) and Bri2 (Itm2b) have been identified as substrates of SPPL2a/b using in vitro overexpression approaches. In agreement with the general concept of RIP it was demonstrated that the TNFα intracellular domain translocates into the nucleus after proteolytic release and influences gene expression thereby inducing the synthesis of the pro-infammatory cytokine IL12. Two of the known substrates suggest a regulatory function of SPPL2a and SPPL2b in the context of the immune system. However, a relevance of these processes and proteolytic events in a complex in vivo system has not been analysed and established yet. It was also suggested that RIPping by SPPL2a and SPPL2b may have more generalized degradative functions beyond mediating reverse signalling by TNFα or other members of this superfamily - a hypothesis being supported by the presence of SPPL orthologues in plants.
Scheme of Proteolysis by SPPL2a/b
Fig. 1: Current model of intramembrane proteolysis by SPPL2a/b.
 
In order to study functions of SPPL2a and SPPL2b in vivo, we have generated mouse lines deficient in either of the two proteases as well as mice being deficient in SPPL2a and -b. Phenotypic analysis of these mice has revealed major immunological abnormalities associated with the deficiency of SPPL2a. These in vivo approaches are combined with biochemical and cell biological experiments studying the differential processing of known and putative substrates in SPPL2a/b deficient cell lines. We are confident, that challenging these mice in infection and inflammation models will contribute to understanding the role of SPPL2a/2b-mediated intramembrane proteolysis in pathophysiology and deepen our understanding of cellular and molecular principles of inflammation. The results of these studies will help us to decide whether SPPL2a and/or -2b might even be considered as potential drug targets and if specific inhibitors of these proteases might be capable of modulating the immune system in a therapeutic way. A central objective of the project is the unbiased search for novel substrates cleaved by SPPL2a and/or –b and to ideally link the biochemical findings with the phenotypes observed in the protease deficient mice.
 
Functional characterisation of novel lysosomal membrane proteins
Lysosomes play a crucial role in the degradation and turnover of different intra- and extracellular macromolecules. Currently, extensive data are available about the proteins of the lysosomal matrix. On the contrary only a minority of the lysosomal membrane proteins has been identified and substantially characterised to date. This contrasts with the number of known and functionally described transport systems or enzymatic activities that have been shown to be associated with this membrane.
Scheme of a Lysosom
Fig. 2: Functions of lysosomal membrane proteins
 
In a previous proteomic analysis of lysosomal membranes we have identified 16 novel enzyme and transporter proteins and 12 novel proteins of unknown functions not previously assigned to lysosomal membranes. Lysosomal localisation of several of these novel proteins could be confirmed by overexpression studies. For two of these novel proteins, "disrupted in renal carcinoma 2" (DIRC2) and "transmembrane protein 192" (TMEM192), we have performed an in-depth biochemical characterisation including validation of lysosomal localisation of the endogenous proteins as well as identification of the sequence-determinants required for lysosomal targeting. DIRC2, a member of the "major facilitator superfamily" was shown to be proteolytically processed by cathepsin L and to exhibit transport activity in a whole-cell electrophysiological assay employing a plasma membrane-localised mutant. TMEM192 a novel protein without any homology to known lysosomal membrane proteins was demonstrated to form dimers mediated by its cytosolic C-terminal tail. Further studies to unravel the molecular functions of these novel lysosomal membrane proteins are ongoing.
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