DFG Research Unit FOR 2290

Understanding Intramembrane Proteolysis

Defining the Repertoire of Substrates and their Molecular Architectures

Speaker: Prof. Dr. Dieter Langosch, Technische Universität München
Deputy speaker: Prof. Dr. Harald Steiner, Ludwig-Maximillians-Universität München








Substrate Recognition

and Cleavage by the Mitochondrial Rhomboid Protease PARL

Marius Lemberg

Cell biology,


Universität zu Köln


Substrate Recognition

and Processing by Signal Peptide Peptidase-like 2 (SPPL2) Family

Regina Fluhrer

Cell biology,


Universität Augsburg


Mechanistic Characterization of Substrate Recognition by γ-Secretase

Stefan Lichtenthaler



DZNE München


Substrate Recruitment

and Cleavage by


Harald Steiner


LMU München


Substrate Transmembrane Helices: Conformational Flexibility and Recognition by an Enzyme

Dieter Langosch


TU München


Functional Role of Transmembrane Domain Interactions and Intramembrane Cleavage

of Microglial Innate Immunity Receptors

Christian Haass


LMU München


Dynamics of



Martin Zacharias


Christina Scharnagl

in silico modeling

TU München


Investigation of the Molecular Dynamics of Substrate Transmembrane α-Helices by Solution and Solid-State NMR Spectroscopy


Claudia Muhle-Goll


Daniel Huster



liquid-state NMR


solid-state NMR



KIT Karlsruhe


Universität Leipzig



Proteomic Platform

Stefan Lichtenthaler


DZNE München


Project P1: Substrate Recognition and Cleavage by the Mitochondrial Rhomboid Protease PARL


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.

Project P2: Substrate Portfolio of the Signal Peptide Peptidase-like 2 (SPPL2) family


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.

Project P3: Identification and mechanistic characterization of a new class of γ-secretase substrates and non-substrates with short extracellular domains


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.


Project P4: Substrate Recognition and Cleavage by γ-Secretase


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.

Project P5: The Conformational Flexibility of Transmembrane Helices in Substrate Recognition and Cleavage


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.


Project P6: Role of transmembrane domain interactions for g-secretase substrate recognition using the TREM2/DAP12 complex


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.

Project P7: Dynamics of Substrate-Protease Interactions


Combining available structural and biochemical data with molecular modeling and simulation methods we will derive structural models in order to advance the understanding of how substrates are recognized and processed by γ-secretase and other intramembrane proteases.


MD simulations and molecular docking will be used to analyze structure, dynamics and enzyme interactions of substrates and to generate putative structural models for substrate binding to γ-secretase. These models will be generated by integrating available experimental data on mutations in the enzyme and substrates and biochemical and biophysical data obtained by experimental FOR2290 groups.


The models will form a basis for understanding the substrate recognition and possible design of inhibitors and modulators.


Project P8: Investigation of Molecular Dynamics of Substrate Transmembrane α-Helices by Solution and Solid-State NMR Spectroscopy


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.


Project P9: Proteomic Platform


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.


Research Unit FOR 2290