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

胰岛素降解酶(IDE，胰岛素)是一种主要的胞内肽酶。 在胰岛素的分解代谢中很重要，淀粉样β多肽，以及可能的其他信号和 胞内肽。因此，它的细胞生理学在治疗中引起了相当大的兴趣。 糖尿病和阿尔茨海默病等疾病的风险。然而，IDE对这些操作的站点 内化到或以其他方式存在于内体系统中的多肽，还有待于 完全理解。我们建议解决这个细胞质酶是如何遇到的问题 底物多肽，如胰岛素，在内体系统中，探索一种新的IDE机制 亚细胞定位到这个隔间。特别是，我们建议将IDE传输到 通过与膜上的阴离子脂类，特别是磷脂酰肌醇，通过一种 聚阴离子结合部位。在第一个目标中，我们将使用IDE多阴离子中心来验证这一假设 突变体，通过改变关键的肌醇磷脂的水平和表达肌醇磷脂 有约束力的竞争者。我们还建议，在第二个目标中，研究 胰岛素和淀粉样蛋白(β(多肽(分解代谢。我们将操控内吞体 利用酶的突变形式降低内体定位的IDE水平，通过 降低PtdIns(3)P水平，并通过将其与PtdIns(3)P- 目标域。在第三个目标中，我们将研究多阴离子依赖的IDE和 它在影响其胞浆多肽分解代谢中的作用。我们将使用IDE突变体来测试效果 用配基捕获技术鉴定的多肽底物的水解性活化和 多肽分析。我们还将测试肌醇磷酸和肌醇对细胞IDE的激活 其他潜在的内源性激活剂。我们将使用我们的诱捕技术来识别其他 内源性效应器。这些研究将更清楚地了解IDE是如何执行其 生理功能，对治疗IDE相关的病理生理状态大有裨益。

Insulin-degrading enzyme (IDE, insulysin) is a primarily cytosolic peptidase shown to be important in the catabolism of insulin, the amyloid beta peptide, and likely other signaling and intracellular peptides. Its cellular physiology is therefore of considerable interest in the treatment of disorders such as diabetes and Alzheimer's disease. However the site of IDE action on these peptides, which are internalized into or otherwise present in the endosomal system, is yet to be fully understood. We propose to address the question of how this cytosolic enzyme encounters substrate peptides, like insulin, in the endosomal system, exploring a novel mechanism for IDE subcellular localization to this compartment. In particular, we propose that IDE is trafficked to endosomes by binding to membrane anionic lipids, particularly phosphoinositides, through a polyanion-binding site. In the first aim, we will test this hypothesis by using IDE polyanion site mutants, by altering levels of a key phosphoinositide, and by expressing a phosphoinositide binding competitor. We further propose to study, in the second aim, the participation of endosomal IDE in insulin and amyloid(beta(peptide(catabolism. We will manipulate endosomal IDE levels using mutant forms of the enzyme with reduced endosome localization, by decreasing PtdIns(3)P levels, and by increasing endosomal IDE by fusing it with a PtdIns(3)P- targeting domain. In the third aim, we will study the polyanion-dependent activation of IDE and its role in affecting its catabolism of cytosolic peptides. We will use IDE mutants to test the effect of activation on hydrolysis of peptide substrates identified using a ligand trapping technique and peptidomic analyses. We will also test for activation of cellular IDE by inositol phosphates and other potential endogenous activators. We will use our trapping technique to identify other endogenous effectors. These studies will develop a clearer picture of how IDE carries out its physiological functions and greatly benefit efforts to treat IDE related pathophysiological states.(