Mechanistic characterization of substrate recognition by gamma-secretase

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This project tests the hypothesis that a new class of unconventional substrates exists for the intramembrane protease gamma-secretase. These new substrates will be used to define the as yet non-understood molecular features, which make a membrane protein a substrate for gamma-secretase. Identifying these features is of central relevance for the research group and will allow us to understand the similarities, but also the differences in substrate recognition and cleavage between the different families of intramembrane proteases. We have discovered an unexpected, novel function for gamma-secretase which is shared with the rhomboid intramembrane protease family, namely the direct shedding of a full-length membrane protein without a prior truncation of the substrates' ectodomain by shedding. We have identified the B cell maturation antigen (BCMA) as a novel substrate. gamma-secretase cleavage results in secretion of the BCMA ectodomain and modulation of BCMA receptor function. In contrast to known gamma-secretase substrates BCMA has a short ectodomain, but our data suggest that additional motifs must exist in the BCMA sequence, which allow BCMA to be directly cleaved by gamma-secretase. Bioinformatically, we have identified 100 additional membrane proteins with a short ectodomain, suggesting that several of them - together with BCMA - form a new, unconventional class of gamma-secretase substrates, while others will be non-substrates and not be cleaved by gamma-secretase. Identification of both sets of proteins (substrates and non-substrates) is essential to understand which features besides the ectodomain length define a membrane protein as a gamma-secretase substrate. The project has three parts. 1. First, we will use an unbiased proteomic approach and a candidate approach to systematically determine which of the 100 membrane proteins with short ectodomains are new substrates, and which are non-substrates for gamma-secretase. 2. Second, we will validate selected gamma-secretase substrates and non-substrates by combining cellular assays and in vitro gamma-secretase assays. 3. Third, we will use domain swap experiments and mechanistically analyze the molecular architecture of gamma-secretase substrates and non-substrates. The project uses state-of-the-art technologies and a close collaboration with other partners in this consortium. As an added value this project has direct implications for deciphering more comprehensively the physiological functions of gamma-secretase, which is also important for drug development targeting gamma-secretase in cancer and neurodegeneration.