The Notch pathway regulates many cellular processes, including development and stem and progenitor cell self-renewal and differentiation. Regulation of Notch signaling is tightly controlled and aberrant Notch activity has pathologic consequences in a wide array of diseases, including cancer and kidney disease. Notch signaling is regulated by a signaling paradigm deemed regulated intramembrane proteolysis (RIP), during which protein substrates undergo ectodomain shedding, followed by cleavage within the lipid bilayer by the ?-secretase protease complex to release intra- and extra-cellular signaling peptides, such as the transcription factor Notch intracellular domain (NICD). ?-Secretase has many substrates and, while the exact mechanisms that regulate its activity level and substrate specificity are still unclear, many proteins have been shown to directly interact with the complex to either enhance or inhibit its cleavage activity. In addition, only a fraction of cellular steady-state ?-secretase complexes are catalytically active and the exact function of the inactive complexes remains unknown. Can RIP be regulated by a tissue- and context-specific modulation of ?-secretase activity? We hypothesize that Notch signaling can be regulated in response to biological environmental stimuli by interaction of ?-secretase with distinct, cell type-specific protein modulators to dynamically regulate the equilibrium between active and inactive iii complexes. I have addressed this research question with two projects focused on the discovery of novel Notch regulators in different tissue and disease models. Hypoxia contributes to the metastatic progression of breast cancer by promoting Notch signaling. We have discovered that the master regulator of hypoxia, Hif-1?, stimulates ?-secretase activity by directly interacting with the ?-secretase complex in a novel, non-canonical mechanism. The breast cancer stem cell (BCSC) population has been proposed to mediate Notch pro-metastatic effects. We hypothesize that Hif-1?-mediated activation ?-secretase/Notch is critical for the regulation of BCSCs, which we have investigated using mammospheres as a model. Our lab has identified a putative, novel endogenous ?-secretase inhibitor in the kidney, gamma-glutamyltransferase (GGT). Notch signaling is critical for kidney development, but is pathogenic if activated in adult kidneys. We hypothesize that GGT regulation of ?-secretase activity acts as a mechanism to repress Notch signaling after kidney development. In this study, we have investigated the mechanism by which GGT inhibits ?-secretase activity in biochemical assays and evaluated if GGT regulates Notch signaling in a biological setting.