Rhomboids are universally conserved intramembrane serine proteases that impact on a variety of important cellular processes.
While analysis of bacterial rhomboids has provided first insights of how cognate transmembrane domains are selected for cleavage, for the more distant mitochondrial rhomboid protease PARL still only very little is known.

Central aim of this project within the FOR2290 is to decipher how PARL selects its substrates. This is of particular relevance since PARL has an active site topology opposite to classical rhomboid proteases in bacteria and the eukaryotic secretory pathway, indicating that it has evolved a unique substrate selection mechanism.

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.

We will now validate the newly identified candidate substrates and determine the precise determinants within these substrates that affect recognition by SPPL2 and processivity. Finally we will investigate the specific prerequisites for substrate recognition within the SPPL2 proteases.

γ-Secretase belongs to the family of intramembrane proteases and has a key function in health and disease. Yet, an in-depth knowledge of the factors governing substrate recognition by γ-secretase is not available.

Addressing this fundamental question is essential to unravel the mechanisms of substrate cleavage, to establish similarities and differences among different intramembrane protease families and for developing substrate-selective protease inhibitors for potential medical use.

Based on our successful work in the first funding period we propose a model for the interaction of γ-secretase with its substrates and their cleavage. Testing this model and thereby unraveling the mechanisms of substrate cleavage is the major aim of our application.

γ-Secretase is a pivotal intramembrane protease and major Alzheimer disease (AD) drug target. Besides the AD-associated β-amyloid precursor protein (APP) substrate C99, the enzyme has about hundred other substrates. Despite successful structure determination of γ-secretase, it is still only poorly understood how its substrates are recognized and selected.

We could already identify the binding sites of C99 at the amino acid level as well as the subunits to which C99 substrate binds. In continuation we will now also determine the precise contact points of C99 at the amino acid level of the protease. Finally, we want to identify sequence determinants of γ-secretase substrates, which are important for recognition and cleavage and which allow distinguishing them from non-substrates. We expect that our continuous studies, in close collaboration with other groups of the FOR2990 network, will reveal fundamental knowledge on how intramembrane proteases recruit and cleave their substrates.

The primary structure of a substrate transmembrane (TM) helix affects its processing by an intramembrane protease. Substrate processing includes multiple levels, such as recognition, translocation, and bond scission. Previous results reveal a complex role of helix flexibility in substrate processing but it remains unclear how exactly the conformational flexibility of a substrate TM helix as well as its mobility in a bilayer are connected to its recognition, uptake, cleavage, and release.

In this project we will systematically assess how site-specific helix flexibility is affected by changing the primary structure of natural and novo designed substrates of γ-secretase, SPPL2 and PARL. Helix flexibility will be related to cleavability performed in other projects of FOR2290. Furthermore, assessing the interactions between TM helices of γ-secretase and various substrates will inform on preferential sites of initial contact.

This project addresses two central questions of this research unit, namely what are the critical requirements for a substrate to be cleaved within its transmembrane (TM) domain and how do TM domain interactions (i.e. dimerization) influence substrate recognition by γ-secretase and other intramembrane cleaving proteases such as members of the SPP/SPPL family.

To identify such properties, we will use the pathologically highly relevant TREM2/DAP12 interaction as a model system. Sequence variants within TREM2 dramatically increase the risk for Alzheimer's disease and related neurodegenerative disorders. TREM2 - a type-1 transmembrane innate immune receptor - is a substrate for regulated intramembrane proteolysis and forms a heteromeric complex with its signaling partner DAP12.

Prof. Martin Zacharias

Technische Universität München

Prof. Daniel Huster

Universität Leipzig

In this project, we investigate the structure and dynamics of transmembrane (TM) proteases’ substrates using a combination of solution and solid-state NMR spectroscopy. As the exact mechanism by which transmembrane proteases recognize their substrates is still not clear, we investigate if the molecular dynamics of a TM helix may determine its suitability as a substrate for intramembrane proteolysis.

To this end, we will apply numerous NMR techniques in solution and in membrane reconstituted systems. In close collaborations with the cell biology and computer simulation groups within the consortium this approach will shed more light on the mechanism by which transmembrane proteases recognize/bind and process their substrates.

The proteomic platform will be an integral part of the research consortium and performs the numerous quantitative mass spectrometry measurements required for projects P1, P2, P3 and P4.

The results of the mass spec analyses will be also relevant for additional projects in the consortium, including the molecular modeling studies.

The platform has two high resolution Orbitrap mass spectrometers (Q Exactive and Q Exactive HF) plus associated equipment, including nanoLCs, and has expertise with the required methods, such as label free quantification and different isotope labelcontaining methods.