Identification of novel genes regulating metalloproteinase activity

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DESCRIPTION (provided by applicant): I am a German MD/Ph.D. clinician-scientist with extensive training in Internal Medicine and Nephrology at Johns Hopkins and Harvard. My research focuses on the epidermal growth factor receptor (EGFR) signaling pathway. In my earlier work I discovered that certain metalloproteinase-cleaved EGF ligands are involved in G-protein signal transduction to MAPkinase and also in osmotic stress signaling to aquaporin water channels. In the kidney, the EGF ligand HB (heparin-binding)-EGF is involved in tubular repair after injury. In the heart it is involved in the generation of cardiac hypertrophy. Ectodomain (ECD) cleavage is also involved in a number of other diseases, like Alzheimer's disease, cancer and inflammation. The signal transduction pathways regulating cleavage are essentially unknown. Metalloproteinase-directed therapies are limited by side effects, or non-existent. With the Whitehead Institute and the Nephrology program at Harvard I have chosen the ideal research and clinical environment to continue to make significant contributions in the field and develop my career as an independent clinician-scientist in Nephrology. The goal of my study is to identify novel genes that regulate ECD cleavage using a high-throughput expression cloning strategy. We detect cleavage of EGF-ligands in a FACS-based assay using cells stably expressing a ligand with an ECD epitope tag and a C-terminal EGFP-fusion. The ECD can be detected with a fluorochrome-coupled anti-epitope antibody, the C-terminus by green fluorescence. In the uncleaved state any given cell has a 1:1 ratio of outside to inside fluorescence, as measured by FACS on life single cells. Stimulation of cleavage decreases, while inhibition of basal or induced cleavage increases this ratio. Infection of a retroviral cDNA library or of a bar-coded shRNAi library, followed by FACS sorting of cells which show altered cleavage ratio allows the identification of novel positive or negative regulators of ECD cleavage. Proof of principle experiments have already been completed. Our long-term goal is the development of new candidate therapies for medically important diseases with a wide impact on the general population. As example, acute kidney failure and heart failure still have a very high mortality despite years of clinical and basic research. Knowledge generated by our research will likely also be extendable to common diseases like cancer, alzheimer's disease or inflammatory disorders.