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

胰岛素降解酶（IDE，胰岛素）是一种主要的胞质肽酶， 在胰岛素、淀粉样β肽和可能的其他信号传导中起重要作用， 胞内肽因此，其细胞生理学在治疗中具有相当大的意义。 糖尿病和老年痴呆症等疾病。然而，IDE对这些网站的操作 被内化或以其他方式存在于内体系统中的肽还有待于研究。 完全理解我们建议解决这个问题，这种胞质酶如何遇到 底物肽，如胰岛素，在内体系统中，探索IDE的新机制 亚细胞定位到这个区室。特别是，我们建议IDE被贩运到 内体通过与膜阴离子脂质，特别是磷酸肌醇结合， 聚阴离子结合位点。在第一个目标中，我们将通过使用IDE聚阴离子位点来检验这一假设 突变体，通过改变关键磷酸肌醇的水平，并通过表达磷酸肌醇 绑定竞争对手。我们还建议，在第二个目标中， 内体IDE在胰岛素和淀粉样蛋白（β-肽（cataline.我们将操纵核内体 IDE水平使用具有减少的内体定位的酶的突变形式，通过 降低PtdIns（3）P水平，并通过将其与PtdIns（3）P- 靶向结构域。在第三个目标中，我们将研究IDE的聚阴离子依赖性激活， 其在影响其对胞浆肽的催化中的作用。我们将使用IDE突变体来测试效果 使用配体捕获技术鉴定的肽底物水解活化， 肽组分析。我们还将测试磷酸肌醇对细胞IDE的激活， 其他潜在的内源性激活剂。我们将使用诱捕技术来识别其他 内源性效应子这些研究将更清楚地了解IDE如何执行其 生理功能，并大大有利于努力治疗IDE相关的病理生理状态。(

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.(