Insulin Degrading Enzyme: Physiological Function and its Spacial and Activity Modulation

This project will focus on the physiological function of IDE in regards to its function as a catabolic enzyme for bioactive peptides and how this function is affected by the intracellular localization of the enzyme. We will study the mechanisms responsible for the localization of IDE to endosomes and other intracellular compartments. The participation of anionic lipids, particularly phosphoinositides, in IDE localization to endosomes will be studied using microscopy and subcellular fractionation. We further propose to study the participation of endosomal IDE in insulin peptide catabolism. We will manipulate endosomal IDE using mutant forms of the enzyme with reduced endosomal binding, by decreasing PtdIns(3)P levels, and by increasing endosomal IDE by fusing it with a PtdIns(3)P-targeting FYVE domain. In the third aim we will study the anion-dependent activation of IDE and its role in affecting the enzymes cellular functions. We will use mutant IDEs to trap and identify endogenous peptide substrates and endogenous effectors. We will test cellular inositol phosphates and any newly identified effectors on IDE activity in cells on IDE activity.