Recent data suggest that intramembrane proteolysis is a surprisingly slow process whose overall kinetics is composed of the kinetics of several steps. These steps may include:
It is currently unclear which of the above step(s) limit the rate of proteolysis, how the ratelimiting step(s) depend on the primary structures and/or the conformational dynamics of the substrates, how disease-related point mutations affect these properties and which step(s) are relevant for substrate/non-substrate discrimination. Furthermore, we do not know whether all intramembrane proteases employ exosites for substrate binding, where they are located, and whether substrates are recognized and/or cleaved as monomers or multimers.
γ-Secretase is a pivotal intramembrane protease and major Alzheimer disease (AD) drug target. Besides the AD-associated β-amyloid precursor protein (APP), currently in the range of hundred substrates are known to be cleaved by γ-secretase. How these are recognized and selected is only poorly understood. Despite recent structural information of γ-secretase, the substrate-binding site(s) of the enzyme are not yet defined. Moreover, it is not known which substrate features make a γ-secretase substrate a “good” (i.e. efficiently cleavable) or a “bad” (i.e. poorly cleavable) one, respectively.
In this project proposal, we will identify the substrate-binding sites of γ-secretase using engineered substrates carrying photocrosslinkable amino acids at defined positions. Thus, upon UV irradiation, the subunits, which directly bind substrate will be unambiguously identified. Using this approach, which will be introduced to our research field for the first time, we will initially start to map the binding sites of APP and then extend this analysis to the crucial γ-secretase substrate Notch1 and other substrates of interest (goal 1).
By mutational analysis, we will additionally identify and define sequence determinants that govern the efficiency of substrate cleavage in proteolysis assays (goal 2).
Finally, we will analyze how the lipid environment of the protease will modulate the substrate recognition process (goal 3).
Our studies will provide fundamental insights into how intramembrane proteases recruit and cleave its substrates.
A primary event in substrate selection is its recognition by the cognate intramembrane protease. We posit that recognition as well as subsequent cleavage of a substrate is crucially influenced by the conformational flexibility of its transmembrane helix, an aspect that is currently unexplored!
In Goal 1, we will investigate transmembrane helix motions in a comparative study of substrates and non-substrates for different intramembrane proteases (γ-secretase, rhomboid PINK1, signal peptide peptidases). To this end, we will apply techniques primarily involving mass spectrometry to study local unfolding around helical cleavage regions as well as global conformational changes of substrate transmembrane helices. Biologically relevant patterns of helix flexibility will be uncovered by comparing the impacts of point mutations on flexibility and on substrate binding and cleavage. Apart from these applied aspects, a systematic comparison of primary structure and transmembrane helix dynamics will yield important new insights into the way both are connected.
In Goal 2, we will develop FRET-based and biochemical assays to study the interaction of the C99 TM domain and the substrate-binding presenilin TM domain in order to identify substrate amino acids critical for binding. Such assays will also enable us to investigate the potential role of the C99 TM domain dimer in substrate/enzyme interaction. Another important question will adress the regulation of substrate/enzyme interaction by lipids by connecting the interaction in different lipid environments to FRET-based analysis of substrate/lipid interaction to be studied in parallel.
This project addresses two central questions of this research unit, namely what defines a substrate to be cleaved within its TM domain and how do TM domain interactions (i.e. dimerization) influence substrate recognition of γ-secretase.
As the major focus of the other participants is on in vitro systems, this project also provides complementary in vivo approaches, which allow to translate and extend data from structural and biochemical analyses in primary glial cells as well as animal models (genome edited zebrafish and mice). We will use the pathologically highly relevant TREM2/DAP12 interactions as an in vivo model to identify such properties and to provide this information for computational and structural analysis for projects 7-8.
In parallel this project may reveal a pathologically and therapeutically highly relevant signaling pathway. Four major points combined in two goals will be specifically investigated