UNIVERSITY OF CHICAGO, THE
Metalloprotease is the most abundant within the five protease classes in humans. Insulin degrading enzyme(IDE) is a zinc-metalloprotease that is involved in the clearance of insulin and amyloid (3 (A3), two keyproteins for the development of diabetes and Alzheimer's disease, respectively. Accumulating geneticevidence strongly suggests that IDE is a potential drug target for type 2 diabetes and Alzheimer's disease. Inorder to develop tools to explore the therapeutic potential of IDE, we have recently solved the x-ray crystalstructures of human IDE in complex with insulin B chain, Ap, amylin, and glucagon at 2.1-2.6A resolution.Our structures reveal a novel mechanism for substrate recognition and control of catalysis of IDE.Specifically, we found that IDE consists of two 56kDa functional N- and C-terminal domains (IDE-N and IDE-C, respectively) and they form an enclosed cage just large enough to encapsulate small peptides such asinsulin. The extensive contacts between IDE-N and IDE-C keep the degradation chamber of IDEinaccessible to substrates. IDE stays in this closed conformation normally and the repositioning of IDEdomains is the key control step in allowing substrate access to the catalytic chamber. The enclosedsubstrate undergoes conformational changes to interact with two discrete regions of IDE for its degradation.In this application, we propose to better understand this intriguing regulation. We will perform mutagenicanalysis to begin to address the opening process as well as determine the structures of two key steps for thecatalytic cycle of IDE, substrate-free IDE closed and open conformations. We will also obtain the structuralbasis in how IDE recognizes disulfide-bond containing IDE substrates and high affinity peptidomimetichydroxamates that can potently inactivate IDE activity. Furthermore, we propose to construct hyperactiveIDE mutants and test their ability to degrade Ap in cultured neuronal cells. Success of these aims will notonly broaden our knowledge in how proteases recognize their substrates and control their proteolytic activitybut also provide valuable information in the future design of IDE-based therapeutics.