Molelcular Dissection of Ectodomain Shedding: Biology of BACE1 and Other Sheddases.

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This Discovery Grant focuses upon our independent, long-term study of sheddases, an important class of enzymes that facilitate "ectodomain shedding" in growth and development. Currently, our model sheddase is BACE1, a type I transmembrane protein related to the pepsin and retroviral aspartic acid protease families. A single transmembrane sequence (TMS) links the ectodomain to the cytosolic tail, constituting a unique feature amongst aspartic proteases. Recent literature has revealed that BACE1 can cleave a more diverse array of substrates than anticipated. Since our recent findings have also revealed that metal ions can interact with BACE1, we hypothesize that copper modulates its physiological properties.***Objectives***Using a variety of biochemical and biophysical tools, this research program examines the diverse physiological roles and molecular mechanisms of sheddases beyond their canonical enzymatic function. For our model system, the goals include characterizing the tertiary and quaternary structure of BACE1, as well as how metal ions affect its function in vitro and in vivo. Specifically, we will: 1) express recombinant BACE1 to identify and characterize its metal ion-binding site(s); 2) determine the 3D structure of two BACE1 constructs (i.e. #1, ectodomain; #2, TMS and cytoplasmic domain) in the absence and presence of copper; and 3) assess the in vivo importance of copper as a BACE1 ligand.****Methodology****Both cellular (HEK293T) and cell-free systems will be used to express our model protein BACE1. Affinity-purified BACE1 will be subjected to limited proteolysis and Mass Spectrometry to identify metal ion-binding residues and induced conformational effects. Subunit counting and FLIM-FRET will be used to assess BACE1 homo-interactions in living cells. Purified BACE1 fragments will be subjected to in vitro structural analyses (NMR and X-ray Crystallography), and BACE1-copper complexes will be examined in vivo (Drosophila, and Long-Evans Cinnamon rats).****Novelty & Impact***There are three innovative technological aspects within this sheddase program. Using BACE1 as our current model, subunit counting will determine its stoichiometry in living cells. Secondly, a novel cell-free protein synthesis system will allow for analyses of the BACE1 TMS. Thirdly, a novel Drosophila model will allow for the study of BACE1-copper complexes in vivo. Given that the structure and multifaceted functions of sheddases remain largely unexplored, the state-of-the-art objectives and methodologies within this research program (and leading-edge grant proposal) provide a new paradigm for characterizing protein interactions in membranes. Uniquely positioning NSERC and its HQP trainees in this fundamental area of ectodomain shedding biology, our detailed analyses of sheddases will help future industrial partners to manipulate their diverse physiological roles.***************