A major aim of our research group is to understand what molecular features qualify a membrane protein as a substrate for intramembrane proteolysis. Answering this question is not only essential to elucidate the physiological function of a protease and evaluate its potential as a drug target but also to understand how a protease recognizes its substrates and cleaves them mechanistically and to elucidate how a protease differs from related proteases. In order to address this question it is necessary to identify the membrane proteins that are cleaved by a protease (substrates) and those membrane proteins that are not cleaved (non-substrates).
Combining methods from biochemistry (such as domain swap experiments, together with P4 and P6), bioinformatics (sequence comparisons, together with P7) and biophysics (analyzing structural parameters, together with P5 and P8) will then allow elucidating the molecular characteristics that qualify a membrane protein as a protease substrate.Among the four major families of intramembrane proteases – rhomboids, SPP/SPPLs, gsecretase and S2Ps – we focus on three for substrate identification and mechanistic analysis, namely PARL, the SPPL2 subfamily and γ-secretase.
Rhomboids are universally conserved serine proteases that impact on a variety of important cellular processes. While analysis of bacterial rhomboids has provided first insights of how cognate substrates are selected, for the more distant mitochondrial rhomboid protease PARL still only very little is known.
Central aims of this project are to identify new PARL substrates and to decipher cleavage determinants. 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 recognition and gating mechanism. Previous work combining classical genetics and substrate candidate testing has revealed several mitochondrial proteins that can be cleaved by PARL, however, the relevance of a number of these reports are currently still under debate. We and others recently reported that the Parkinson’s disease-associated protein kinase PINK1 is a physiological PARL substrat. Intriguingly, we observed that disease-associated mutations and replacement of conserved helix-destabilizing residues interfere with PARL-catalyzed processing. Since related features exist in several other intramembrane protease substrates, this suggests that there are common principles for proteolysis within the membrane. Here we propose to systematically define the physiological substrate spectrum of PARL in tissue culture cells and to decipher its specificity in a cell-free system.
Specific goals are:
To this end, we plan to develop an in vitro assay for detergent-solubilized and purified PARL enabling detailed kinetic analysis analyzing the role of individual substrate determinants. Identification of cleavage sites of physiological as well as artificial PARL substrates by proteomic methods will provide a valuable resource to determine its cleavage site consensus and to develop a prediction algorithm. Combined with biophysical analysis performed within this consortium, this enhanced sequence analysis likely will provide an understanding of how transmembrane helix dynamics impact on PARL-catalyzed cleavage.
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 proteases. Using candidate approaches so far, only six substrates for the SPPL2 sub-family have been identified.
Goal 1: Project 2, thus aims to expand the spectrum of known SPPL2 substrates using proteomic methods and to elucidate the common determinants of newly identified and known SPPL2 substrates. With this, project 2 contributes significantly to the systematic identification of novel substrates for intramembrane proteases, one major aim of the research group.
Goal 2: By determining the cleavage sites in known and newly identified SPPL2 substrates, this project will deliver the basis to identify further candidate substrates using computational analysis. With the development of robust cell free in vitro assays, we provide an important tool to perform kinetic studies on the respective SPPL2 family members.
Goal 3: To further expand the knowledge of how members of the SPPL2 family recognize their substrates and which intrinsic structural determinants of a substrate are required for efficient proteolysis, we aim to investigate proteolysis of TNFa by SPPL2a and SPPL2b in more detail. In particular, we will investigate the influence of certain amino acids, for instance potentially helix destabilizing amino acids such as b-branched amino acids, within the transmembrane (TM) domain of TNFa as well as the role of TNFa-palmitoylation on substrate turnover by SPPL2a/b intramembrane proteases. In the future, we will expand these analyses to other known and newly identified substrates.
The results of this project will further complement the overall aim of the research group, namely to uncover the structural requirements for the proteolytic processing of substrate TM domains and will help to establish computational screening algorithms allowing the prediction of further substrates and common substrate determinates for intramembrane proteases.
Based on our identification of BCMA as a novel, unconventional γ−secretase substrate, we will pursue three goals to address the following two questions: What makes a membrane protein a substrate for γ− secretase? And does γ− secretase share functional and mechanistic characteristics with other intramembrane protease families?
Goal1: We will test the hypothesis that a new class of unconventional substrates exists, where γ−secretase does not only mediate intramembrane proteolysis, but also shedding of the ectodomain.
This will be tested using an unbiased proteomic approach and a candidate approach to determine which of 100 membrane proteins with short ectodomains are new substrates, and which are
non-substrates for γ−secretase. Identification of both sets of proteins (substrates and non-substrates) is essential to understand the molecular architecture of γ−secretase substrates in Goal
Goal2: We will validate selected γ−secretase substrates and non-substrates identified in Goal1 or already identified in our previous experiments. Validation will be achieved combining in vitro γ−secretase assays and cellular assays using FACS analysis and immunoblots.
Goal3: We will mechanistically analyze the molecular architecture of γ−secretase substrates and non-substrates. This includes domain swap experiments between selected substrates and nonsubstrates (with a particular focus on the transmembrane and juxtamembrane domains) as well as alterations of the length of the ectodomain. This mutational analysis will be supported by bioinformatics to identify sequence-based or structural features shared by substrates versus non-substrates. Specifically, we will test the hypothesis that sequence and structure motifs of the juxtamembrane and transmembrane domains are determinants for cleavage by γ−secretase. For substrates the cleavage sites will be determined using mass spectrometry to determine whether their cleavage mechanism is similar or distinct from the traditional, longer γ−secretase substrates. For the validated short γ−secretase substrate BCMA we will also determine whether ligand-binding is a new mechanism directly controlling γ−secretase cleavage of this cell surface receptor.