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PubMed2001

Clearing the brain's amyloid cobwebs.

Elevated cerebral levels of amyloid beta-protein occur universally in Alzheimer's disease, yet only a few patients show evidence of increased Abeta production. Therefore, defects in proteases that degrade Abeta could underlie some or many cases of familial and sporadic AD. This previously neglected topic has begun receiving serious attention. Understanding how proteolysis regulates Abeta levels in the cerebral cortex has implications for both the pathogenesis and the treatment of this protean disorder.

Alzheimer Disease↗
PubMed2003

Enhanced proteolysis of beta-amyloid in APP transgenic mice prevents plaque formation, secondary pathology, and premature death.

Converging evidence suggests that the accumulation of cerebral amyloid beta-protein (Abeta) in Alzheimer's disease (AD) reflects an imbalance between the production and degradation of this self-aggregating peptide. Upregulation of proteases that degrade Abeta thus represents a novel therapeutic approach to lowering steady-state Abeta levels, but the consequences of sustained upregulation in vivo have not been studied. Here we show that transgenic overexpression of insulin-degrading enzyme (IDE) or neprilysin (NEP) in neurons significantly reduces brain Abeta levels, retards or completely prevents amyloid plaque formation and its associated cytopathology, and rescues the premature lethality present in amyloid precursor protein (APP) transgenic mice. Our findings demonstrate that chronic upregulation of Abeta-degrading proteases represents an efficacious therapeutic approach to combating Alzheimer-type pathology in vivo.

Amyloid beta-Peptides↗
PubMed2002

Subcellular localization of Axl1, the cell type-specific regulator of polarity.

Bud-site selection in yeast offers an attractive system for studying cell polarity and asymmetric division. Haploids divide in an axial pattern, whereas diploids divide in a bipolar pattern. AXL1 is expressed in haploids but not diploids, and ectopic expression of AXL1 in diploids converts their bipolar budding pattern to an axial pattern. How Axl1 acts as a switch between the bipolar and axial patterns is not understood. Here we report that Axl1 localizes to the mother-bud neck and division site remnants of haploids. Axl1 is absent from diploids. Axl1 colocalizes with Bud3, Bud4, and Bud10, components of the axial landmark structure. This localization suggests that Axl1 couples the axial landmark with downstream polarity establishment factors. Consistent with such a role, Axl1 associated biochemically with Bud4 and Bud5. Genetic evidence suggests that Axl1 works with Bud3 and Bud4 to promote the activity of the Bud10 membrane protein. Given Axl1's suggested role in morphogenesis and cell fusion during mating, we also examined its localization during this process. Axl1 redistributes independently of the axial landmark to a tight cell surface dot at the tip of each mating projection. These dots are rapidly lost as prezygotes form.

Cell Division↗
PubMed2001

Insulin-degrading enzyme: embarking on amyloid destruction.

Several human disorders are caused by or associated with the deposition of protein aggregates known as amyloid fibrils. Despite the lack of sequence homology among amyloidogenic proteins, all amyloid fibrils share a common morphology, are insoluble under physiological conditions and are resistant to proteolytic degradation. Because amyloidogenic proteins are being produced continuously, eukaryotic organisms must have developed a form of proteolytic machinery capable of controlling these aggregation-prone species before their fibrillization. This article suggests that an intracellular metalloprotease called insulin-degrading enzyme (IDE) is responsible for the elimination of proteins with amyloidogenic potential and proposes a mechanism for the selectivity of the enzyme. In this respect, IDE can also be referred to as ADE: amyloid-degrading enzyme.

Amino Acid Motifs↗
PubMed2003

Does diabetes protect or provoke Alzheimer's disease? Insights into the pathobiology and future treatment of Alzheimer's disease.

Diabetes mellitus has long been considered a risk factor for the development of vascular dementia. Epidemiologic evidence has suggested that diabetes mellitus significantly increases risk for the development of Alzheimer's disease, independent of vascular risk factors. As insulin's role as a neuromodulator in the brain has been described, its significance for AD has also emerged. Insulin dysregulation may contribute to AD pathology through several mechanisms including decreased cortical glucose utilization particularly in the hippocampus and entorhinal cortex; increased oxidative stress through the formation of advanced glycation end-products; increased Tau phosphorylation and neurofibrillary tangle formation; increased b-amyloid aggregation through inhibition of insulin-degrading enzyme. Future treatment of AD might involve pharmacologic and dietary manipulations of insulin and glucose regulation.

Aged↗
PubMed1992

Rat insulin-degrading enzyme: cleavage pattern of the natriuretic peptide hormones ANP, BNP, and CNP revealed by HPLC and mass spectrometry.

The degradation of atrial natriuretic peptide (ANP), brain natriuretic peptide (BNP), and C-type natriuretic peptide (CNP) by insulin-degrading enzyme (IDE) has been investigated. As revealed by high-performance liquid chromatography, all three peptides are sequentially cleaved at a limited number of sites, the latter of which were identified by mass spectrometric analyses. The studies revealed that ANP is preferred as substrate over BNP and CNP. ANP degradation is rapidly initiated by hydrolysis at the Ser25-Phe26 bond. Three additional cleavage sites were identified in ANP after prolonged incubation with IDE; in contrast, three and two bonds were hydrolyzed in BNP and CNP, respectively. Analysis of the nine cleavage sites shows a preference for basic or hydrophobic amino acid residues on the carboxyl side of a cleaved peptide bond. In contrast to most of the peptide fragments generated by IDE activity, the initial ANP cleavage product, F-R-Y, is rapidly degraded further by cleavage of the R-Y bond. Cross-linking studies with 125I-ANP in the presence of sulfhydryl-modifying agent indicate that IDE activity is inhibited at the level of initial substrate binding whereas metal-ion chelating agents only prevent hydrolysis. On the basis of its structural and enzymatic properties, IDE exhibits striking similarity to a number of recently-described endopeptidases.

Amino Acid Sequence↗
PubMed1989

Drosophila insulin degrading enzyme and rat skeletal muscle insulin protease cleave insulin at similar sites.

Insulin degradation is an integral part of the cellular action of insulin. Recent evidence suggests that the enzyme insulin protease is involved in the degradation of insulin in mammalian tissues. Drosophila, which has insulin-like hormones and insulin receptor homologues, also expresses an insulin degrading enzyme with properties that are very similar to those of mammalian insulin protease. In the present study, the insulin cleavage products generated by the Drosophila insulin degrading enzyme were identified and compared with the products generated by the mammalian insulin protease. Both purified enzymes were incubated with porcine insulin specifically labeled with 125I on either the A19 or B26 position, and the degradation products were analyzed by HPLC before and after sulfitolysis. Isolation and sequencing of the cleavage products indicated that both enzymes cleave the A chain of intact insulin at identical sites between residues A13 and A14 and A14 and A15. Sequencing of the B chain fragments demonstrated that the Drosophila enzyme cleaves the B chain of insulin at four sites between residues B10 and B11, B14 and B15, B16 and B17, and B25 and B26. These cleavage sites correspond to four of the seven cleavage sites generated by the mammalian insulin protease. These results demonstrate that all the insulin cleavage sites generated by the Drosophila insulin degrading enzyme are shared in common with the mammalian insulin protease. These data support the hypothesis that there is evolutionary conservation of the insulin degrading enzyme and further suggest that this enzyme plays an important role in cellular function.

Animals↗
PubMed1990

Identification of residues in the insulin molecule important for binding to insulin-degrading enzyme.

Insulin-degrading enzyme (IDE) hydrolyzes insulin at a limited number of sites. Although the positions of these cleavages are known, the residues of insulin important in its binding to IDE have not been defined. To this end, we have studied the binding of a variety of insulin analogues to the protease in a solid-phase binding assay using immunoimmobilized IDE. Since IDE binds insulin with 600-fold greater affinity than it does insulin-like growth factor I (25 nM and approximately 16,000 nM, respectively), the first set of analogues studied were hybrid molecules of insulin and IGF I. IGF I mutants [insB1-17,17-70]IGF I, [Tyr55,Gln56]IGF I, and [Phe23,Phe24,Tyr25]IGF I have been synthesized and share the property of having insulin-like amino acids at positions corresponding to primary sites of cleavage of insulin by IDE. Whereas the first two exhibit affinities for IDE similar to that of wild type IGF I, the [Phe23,Phe24,Tyr25]IGF I analogue has a 32-fold greater affinity for the immobilized enzyme. Replacement of Phe-23 by Ser eliminates this increase. Removal of the eight amino acid D-chain region of IGF I (which has been predicted to interfere with binding to the 23-25 region) results in a 25-fold increase in affinity for IDE, confirming the importance of residues 23-25 in the high-affinity recognition of IDE. A similar role for the corresponding (B24-26) residues of insulin is supported by the use of site-directed mutant and semisynthetic insulin analogues. Insulin mutants [B25-Asp]insulin and [B25-His]insulin display 16- and 20-fold decreases in IDE affinity versus wild-type insulin.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed2005

Alternative splicing of human insulin-degrading enzyme yields a novel isoform with a decreased ability to degrade insulin and amyloid beta-protein.

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.

Alternative Splicing↗
PubMed1996

Identification of gamma-endorphin-generating enzyme as insulin-degrading enzyme.

The EL-4 thymoma cell line contains a peptidase which converts beta-endorphin to beta-endorphin 1-17 (gamma-endorphin), beta-endorphin 1-18, and their corresponding C-terminal fragments. This enzyme was purified approximately 700-fold to a single band on an SDS-polyacrylamide gel (106 kDa) in 16% yield. Estimation of the native molecular weight by molecular sieve chromatography gave a value of approximately 220 kDa, indicating that this enzyme is a dimer. Peptide sequencing demonstrated this activity can be attributed to insulin degrading enzyme, a previously described member of the inverzincin family (Hooper, 1994). Kinetic studies with a number of peptide substrates indicate that the enzyme preferentially cleaves on the amino side of hydrophobic or basic residues. However, the substrate specificity is more complex since not all basic and hydrophobic residues in a peptide are cleaved. The enzyme exhibits a requirement for a P'2 residue. On the basis of kcat/K(m) values, insulin, growth hormone releasing factor, and beta-endorphin are nearly equivalent substrates for the enzyme; however, growth hormone releasing factor and beta-endorphin exhibit a 40-fold higher kcat, but a 10-fold decreased affinity relative to insulin. A role for insulin-degrading enzyme as both a beta-endorphin-processing and -inactivating enzyme is implicated from these studies.

Amino Acid Sequence↗
PubMed1997

Immunohistochemical localization of insulin-degrading enzyme along the rat intestine, in the human colon adenocarcinoma cell line (Caco-2), and in human ileum.

Insulin-degrading enzyme (IDE) has been implicated in the intracellular degradation of insulin in insulin target cells. Knowledge of the existence of this enzyme in the intestine will be beneficial to the achievement of clinical oral efficacy of insulin. A comparative study was conducted with rat intestine, human colon adenocarcinoma (Caco-2) cells, and human ileum. Confocal microscopy analysis using the anti-IDE antibody showed that IDE was localized in the mucosal cells of rat and human intestines, as well as in Caco-2 cells. Immunostaining of this enzyme was homogeneous throughout the cell excluding nucleus, indicating a typical cytosolic distribution in rat and human enterocytes and in Caco-2 cells.

Adenocarcinoma↗
PubMed2000

Degradation of soluble amyloid beta-peptides 1-40, 1-42, and the Dutch variant 1-40Q by insulin degrading enzyme from Alzheimer disease and control brains.

Insulin degrading enzyme (IDE) is a metalloprotease that has been involved in amyloid beta peptide (A(beta)) degradation in the brain. We analyzed the ability of human brain soluble fraction to degrade A(beta) analogs 1-40, 1-42 and the Dutch variant 1-40Q at physiological concentrations (1 nM). The rate of synthetic 125I-A(beta) degradation was similar among the A(beta) analogs, as demonstrated by trichloroacetic acid precipitation and SDS-PAGE. A 110 kDa protein, corresponding to the molecular mass of IDE, was affinity labeled with either 125I-insulin, 125I-Abeta 1-40 or 125I-A(beta) 1-42 and both A(beta) degradation and cross-linking were specifically inhibited by an excess of each peptide. Sensitivity to inhibitors was consistent with the reported inhibitor profile of IDE. Taken together, these results suggested that the degradation of A(beta) analogs was due to IDE or a closely related protease. The apparent Km, as determined using partially purified IDE from rat liver, were 2.2 +/- 0.4, 2.0 +/- 0.1 and 2.3 +/- 0.3 microM for A(beta) 1-40, A(beta) 1-42 and A(beta) 1-40Q, respectively. Comparison of IDE activity from seven AD brain cytosolic fractions and six age-matched controls revealed a significant decrease in A(beta) degrading activity in the first group, supporting the hypothesis that a reduced IDE activity may contribute to A(beta) accumulation in the brain.

Alzheimer Disease↗
PubMed2001

Amyloid beta peptide processing, insulin degrading enzyme, and butyrylcholinesterase.

Amyloid beta peptide implicated in Alzheimers disease is cleaved by insulin degrading enzyme (IDE). Abnormal cholinesterases similar to butyrylcholinesterase (BChE) are found in Alzheimer brain. The similarities between IDE and BChE (which is known to have an arylacylamidase and a metallocarboxypeptidase-like activity) such as their zinc metalloenzyme nature, their localization in glia and their ability to bind amyloid peptide in Alzheimers disease raise interesting questions.

Alzheimer Disease↗
PubMed1995

Insulin-degrading enzyme in a human colon adenocarcinoma cell line (Caco-2).

The activity of insulin-degrading enzyme (IDE), a thiol metalloprotease degrading insulin in many insulin target cells, was determined in human colon adenocarcinoma (Caco-2) cells. Insulin-degrading activity was localized in the cytosol of Caco-2 cells, accounting for 88% of total activity. Western blots and immunoprecipitation showed that IDE was present in the cytosol of Caco-2 cells and contributed to more than 93% cytosolic insulin-degrading activity. Cytosolic insulin degradation was strongly inhibited by IDE inhibitors, including N-ethylmaleimide, 1,10-phenanthroline, p-chloromericuribenzoate, and EDTA, but was not significantly or not as extensively inhibited by strong inhibitors of proteasome, i.e., chymostatin, soybean trypsin inhibitor, leupeptin, and Dip-F. These results suggest that IDE is present in Caco-2 cells, that Caco-2 IDE has properties similar to those of its counterparts in insulin-target tissues, and that it significantly contributes to intracellular insulin degradation.

Adenocarcinoma↗
PubMed1995

Transepithelial transport of insulin: I. Insulin degradation by insulin-degrading enzyme in small intestinal epithelium.

PURPOSE: The purpose of this study was to determine the existence of insulin-degrading enzyme (EC 3.4.22.11) (IDE) in rat intestinal enterocytes. METHODS: Subcellular fractionation, biochemical characterization, immunoprecipitation, and western blots were employed. RESULTS: Insulin-degrading activity was localized in the cytosol, constituting 92% of total insulin-degrading activity. Cytosolic insulin-degrading activity had a pH optimum of 7.5, was almost completely inhibited by IDE inhibitors (N-ethylmaleimide, 1,10-phenanthroline, EDTA, p-chloromercuribenzoate, bacitracin), but was not or only weakly inhibited by others (aprotinin, chymostatin, leupeptin, and diisopropyl phosphofluoridate.) Further, cytosolic insulin-degrading activity had a Km of 78 nM, sharing a similar Km value with insulin-degrading enzyme in non-purified forms. Approximately, 87 +/- 1.7% of cytosolic insulin-degrading activity was removed by the monoclonal antibody to IDE. On the SDS gel, the molecular weight of cytosolic IDE was 110 KD which is the same as that of human IDE. CONCLUSIONS: IDE is the major enzyme which degrades insulin in enterocytes.

Animals↗
PubMed1997

Degradation of Alzheimer's beta-amyloid protein by human and rat brain peptidases: involvement of insulin-degrading enzyme.

We examined the degradation of Alzheimer's beta-amyloid protein (1-40) by soluble and synaptic membrane fractions from post mortem human and fresh rat brain using HPLC. Most of the activity at neutral pH was in the soluble fraction. The activity was thiol and metal dependent, with a similar inhibition profile to insulin-degrading enzyme. Immunoprecipitation of insulin-degrading enzyme from the human soluble fraction using a monoclonal antibody removed over 85% of the beta-amyloid protein degrading activity. Thus insulin-degrading enzyme is the main soluble beta-amyloid degrading enzyme at neutral pH in human brain. The highest beta-amyloid protein degrading activity in the soluble fractions occurred between pH 4-5, and this activity was inhibited by pepstatin, implicating an aspartyl protease. Synaptic membranes had much lower beta-amyloid protein degrading activity than the soluble fraction. EDTA (2mM) caused over 85% inhibition of the degrading activity but inhibitors of endopeptidases -24.11, -24.15, -24.16, angiotensin converting enzyme, aminopeptidases, and carboxypeptidases had little or no effect.

Alzheimer Disease↗
PubMed2002

Naturally secreted oligomers of amyloid beta protein potently inhibit hippocampal long-term potentiation in vivo.

Although extensive data support a central pathogenic role for amyloid beta protein (Abeta) in Alzheimer's disease, the amyloid hypothesis remains controversial, in part because a specific neurotoxic species of Abeta and the nature of its effects on synaptic function have not been defined in vivo. Here we report that natural oligomers of human Abeta are formed soon after generation of the peptide within specific intracellular vesicles and are subsequently secreted from the cell. Cerebral microinjection of cell medium containing these oligomers and abundant Abeta monomers but no amyloid fibrils markedly inhibited hippocampal long-term potentiation (LTP) in rats in vivo. Immunodepletion from the medium of all Abeta species completely abrogated this effect. Pretreatment of the medium with insulin-degrading enzyme, which degrades Abeta monomers but not oligomers, did not prevent the inhibition of LTP. Therefore, Abeta oligomers, in the absence of monomers and amyloid fibrils, disrupted synaptic plasticity in vivo at concentrations found in human brain and cerebrospinal fluid. Finally, treatment of cells with gamma-secretase inhibitors prevented oligomer formation at doses that allowed appreciable monomer production, and such medium no longer disrupted LTP, indicating that synaptotoxic Abeta oligomers can be targeted therapeutically.

Alzheimer Disease↗