Farris Wesley, Leissring Malcolm A, Hemming Matthew L, Chang Alice Y, Selkoe Dennis J
Center for Neurologic Diseases, Department of Neurology, Brigham and Women's Hospital, Harvard Medical School, Boston, Massachusetts 02115, USA.
Biochemistry. 2005 May 3;44(17):6513-25. doi: 10.1021/bi0476578.
Deletion of insulin-degrading enzyme (IDE) in mice causes accumulation of cerebral amyloid beta-protein (Abeta), hyperinsulinemia, and glucose intolerance. Together with genetic linkage and allelic association of IDE to Alzheimer's disease (AD) and type 2 diabetes mellitus (DM2), these findings suggest that IDE hypofunction could mediate human disease. To date, no coding mutations have been found in the canonical isoform of IDE, suggesting that pathological mutations could exist in undiscovered exons or regulatory regions, including untranslated regions (UTRs). However, neither isoforms arising from alternative splicing nor the UTRs have been described. Here, we systematically characterize human IDE mRNAs, identify a novel splice form, and compare its subcellular distribution, kinetic properties, and ability to degrade Abeta to the known isoform. Six distinct human IDE transcripts were identified, with most of the variance attributable to alternative polyadenylation sites. In the novel spliceoform, an exon we designate "15b" replaces the canonical exon "15a", and the resultant variant is widely expressed. Subcellular fractionation, immunofluorescent confocal microscopy, and immunogold-electron microscopy reveal that the 15b-IDE protein occurs in both cytosol and mitochondria. Organelle targeting of both isoforms is determined by which of two translation start sites is used, and only those isoforms utilizing the second site regulate levels of secreted Abeta. 15b-IDE can exist as a heterodimer with the 15a isoform or as a homodimer. The apparent K(m) values of recombinant 15b-IDE for both insulin and Abeta are significantly higher and the k(cat) and catalytic efficiency markedly lower than those of 15a-IDE. In accord, cells coexpressing beta-amyloid precursor protein (APP) and 15b-IDE accumulated significantly more Abeta in their media than those expressing APP and 15a-IDE. Our results identify a novel, catalytically inefficient form of IDE expressed in brain and non-neural tissues and recommend novel regions of the IDE gene in which to search for mutations predisposing patients to AD and DM2.
在小鼠中删除胰岛素降解酶（IDE）会导致脑淀粉样β蛋白（Aβ）积累、高胰岛素血症和葡萄糖不耐受。连同IDE与阿尔茨海默病（AD）和2型糖尿病（DM2）的遗传连锁及等位基因关联，这些发现提示IDE功能减退可能介导人类疾病。迄今为止，尚未在IDE的典型同工型中发现编码突变，这表明病理突变可能存在于未被发现的外显子或调控区域，包括非翻译区（UTR）。然而，尚未描述由可变剪接产生的同工型或UTR。在此，我们系统地表征人IDE mRNA，鉴定一种新型剪接形式，并将其亚细胞分布、动力学特性及降解Aβ的能力与已知同工型进行比较。鉴定出六种不同的人IDE转录本，大部分差异归因于可变聚腺苷酸化位点。在新型剪接异构体中，一个我们命名为“15b”的外显子取代了典型外显子“15a”，并且产生的变体广泛表达。亚细胞分级分离、免疫荧光共聚焦显微镜和免疫金电子显微镜显示15b-IDE蛋白存在于细胞质和线粒体中。两种同工型的细胞器靶向由使用两个翻译起始位点中的哪一个决定，并且只有那些利用第二个位点的同工型调节分泌型Aβ的水平。15b-IDE可以与15a同工型形成异二聚体或作为同二聚体存在。重组15b-IDE对胰岛素和Aβ的表观K(m)值显著更高，而k(cat)和催化效率明显低于15a-IDE。与此一致，共表达β淀粉样前体蛋白（APP）和15b-IDE的细胞在其培养基中积累的Aβ比表达APP和15a-IDE的细胞显著更多。我们的结果鉴定出一种在脑和非神经组织中表达的新型、催化效率低下的IDE形式，并推荐在IDE基因的新区域中寻找使患者易患AD和DM2的突变。
