Alternative Splicing of Human Insulin-Degrading Enzyme Yields a Novel Isoform with a Decreased Ability To Degrade Insulin and Amyloid 
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- 作者：Wesley Farris ; Malcolm A. Leissring ; Matthew L. Hemming ; Alice Y. Chang ; and Dennis J. Selkoe
- 刊名：Biochemistry
- 出版年：2005
- 出版时间：May 3, 2005
- 年：2005
- 卷：44
- 期：17
- 页码：6513 - 6525
- 全文大小：1044K
- 年卷期：v.44,no.17(May 3, 2005)
- ISSN：1520-4995
文摘
Deletion of insulin-degrading enzyme (IDE) in mice causes accumulation of cerebral amyloid
-protein (A), hyperinsulinemia, and glucose intolerance. Together with genetic linkage and allelicassociation of IDE to Alzheimer's disease (AD) and type 2 diabetes mellitus (DM2), these findings suggestthat IDE hypofunction could mediate human disease. To date, no coding mutations have been found inthe canonical isoform of IDE, suggesting that pathological mutations could exist in undiscovered exonsor regulatory regions, including untranslated regions (UTRs). However, neither isoforms arising fromalternative splicing nor the UTRs have been described. Here, we systematically characterize human IDEmRNAs, identify a novel splice form, and compare its subcellular distribution, kinetic properties, andability to degrade A to the known isoform. Six distinct human IDE transcripts were identified, withmost of the variance attributable to alternative polyadenylation sites. In the novel spliceoform, an exonwe designate "15b" replaces the canonical exon "15a", and the resultant variant is widely expressed.Subcellular fractionation, immunofluorescent confocal microscopy, and immunogold-electron microscopyreveal that the 15b-IDE protein occurs in both cytosol and mitochondria. Organelle targeting of bothisoforms is determined by which of two translation start sites is used, and only those isoforms utilizingthe second site regulate levels of secreted A. 15b-IDE can exist as a heterodimer with the 15a isoformor as a homodimer. The apparent Km values of recombinant 15b-IDE for both insulin and A aresignificantly higher and the kcat and catalytic efficiency markedly lower than those of 15a-IDE. In accord,cells coexpressing -amyloid precursor protein (APP) and 15b-IDE accumulated significantly more Ain their media than those expressing APP and 15a-IDE. Our results identify a novel, catalytically inefficientform of IDE expressed in brain and non-neural tissues and recommend novel regions of the IDE gene inwhich to search for mutations predisposing patients to AD and DM2.
-protein (A), hyperinsulinemia, and glucose intolerance. Together with genetic linkage and allelicassociation of IDE to Alzheimer's disease (AD) and type 2 diabetes mellitus (DM2), these findings suggestthat IDE hypofunction could mediate human disease. To date, no coding mutations have been found inthe canonical isoform of IDE, suggesting that pathological mutations could exist in undiscovered exonsor regulatory regions, including untranslated regions (UTRs). However, neither isoforms arising fromalternative splicing nor the UTRs have been described. Here, we systematically characterize human IDEmRNAs, identify a novel splice form, and compare its subcellular distribution, kinetic properties, andability to degrade A to the known isoform. Six distinct human IDE transcripts were identified, withmost of the variance attributable to alternative polyadenylation sites. In the novel spliceoform, an exonwe designate "15b" replaces the canonical exon "15a", and the resultant variant is widely expressed.Subcellular fractionation, immunofluorescent confocal microscopy, and immunogold-electron microscopyreveal that the 15b-IDE protein occurs in both cytosol and mitochondria. Organelle targeting of bothisoforms is determined by which of two translation start sites is used, and only those isoforms utilizingthe second site regulate levels of secreted A. 15b-IDE can exist as a heterodimer with the 15a isoformor as a homodimer. The apparent Km values of recombinant 15b-IDE for both insulin and A aresignificantly higher and the kcat and catalytic efficiency markedly lower than those of 15a-IDE. In accord,cells coexpressing -amyloid precursor protein (APP) and 15b-IDE accumulated significantly more Ain their media than those expressing APP and 15a-IDE. Our results identify a novel, catalytically inefficientform of IDE expressed in brain and non-neural tissues and recommend novel regions of the IDE gene inwhich to search for mutations predisposing patients to AD and DM2.