Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:3.4.24.56 (insulin-degrading enzyme)
 737 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Previous reports demonstrated that insulin is translocated through the cytoplasm to the nucleus of H35 hepatoma cells and suggested that nuclear insulin may be involved in stimulating transcription of immediate-early genes. In a recent study, inhibition of insulin-degrading enzyme with 1,10-phenanthroline, a Zn2+ chelator, caused a significant increase in the nuclear accumulation of insulin. The present study characterized the effects of 1,10-phenanthroline and its nonchelating isomer, 1,7-phenanthroline, on insulin degradation, nuclear accumulation, and stimulation of immediate-early gene expression. 1,10- but not 1,7-phenanthroline inhibited insulin degradation and increased nuclear accumulation of insulin in a dose-dependent manner. 1,7-phenanthroline caused a dose-dependent decrease in the expression of insulin-stimulated immediate-early genes, but had no significant effect on alpha-tubulin mRNA levels. In the presence of insulin, Northern analysis revealed that 1,10-phenanthroline at all concentrations tested increased alpha-tubulin mRNA levels, but had a biphasic effect on insulin-stimulated immediate-early gene expression. At low concentrations (5-200 microM), 1,10-phenanthroline increased the expression of insulin-stimulated g33, c-fos, and Egr-1 mRNA. At concentrations greater than 1 mM, insulin-stimulated immediate-early gene expression was decreased similar to the effect seen with 1,7-phenanthroline. Nuclear run-on analysis demonstrated that high concentrations of 1,10-phenanthroline decreased insulin-stimulated immediate-early gene transcription but had no effect on transcription of alpha-tubulin. However, low concentrations of 1,10-phenanthroline did not increase transcription of any genes.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:1,10-Phenanthroline increases nuclear accumulation of insulin in response to inhibiting insulin degradation but has a biphasic effect on insulin's ability to increase mRNA levels. 802 92

Interest in tick-borne pathogens has been enhanced by the emergence of Lyme disease and, more recently, human and animal ehrlichioses. In order to facilitate investigations of the vector phase of tick-borne disease agents in vitro, several new cell lines derived from embryonated eggs of northern (IDE lines) and southern (ISE lines) populations of the tick Ixodes scapularis were developed. The establishment and characteristics of 4 IDE (IDE1, 2, 8, and 12) and 2 ISE (ISE5 and 18) lines were described. Primary cultures were initiated in L-15B medium at 31 C from a single egg mass each and established lines developed a morphologically distinct phenotype. Myoblasts were present during the first year after isolation in several lines as isolated clusters or sheets covering the whole flask. Cell line extracts resolved by isoelectric focusing were characterized for 3 isozymes (lactate dehydrogenase, malate dehydrogenase, and malic enzyme). The combined banding patterns allowed discrimination between Ixodes cell lines and a Rhipicephalus appendiculatus cell line. Two lines, i.e., ISE5 and ISE18, had unique isozyme bands. Chromosome numbers and morphology conformed to those described from tissue squashes of I. scapularis.
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PMID:Establishment, maintenance and description of cell lines from the tick Ixodes scapularis. 806 20

Insulin degrading enzyme (IDE) is an evolutionarily conserved, nonlysosomal metalloprotease that has been implicated in the cellular degradation and processing of insulin. However, the site and the mode of the action of this enzyme are unclear. We have addressed these questions by establishing several Ltk- cell lines that can overexpress human insulin-degrading enzyme (hIDE) upon glucocorticoid induction. The level of overexpression of hIDE protein and transcripts in these lines correlates well with an increase in insulin degradation in both cell lysates and intact cells. Comparison of the deduced amino acid sequences of mammalian and Drosophila IDEs reveals a conserved carboxyl-terminal peroxisomal targeting sequence (A/S-K-L), suggesting that IDE may be localized in peroxisomes. To test this possibility, we determined the cellular location of the stably transfected hIDE by both immunofluorescence and immunocryoelectron microscopy. The overexpressed hIDE predominantly colocalized with catalase in peroxisomes, although IDE was also found in the cytosol at a much lower concentration. These results demonstrate that stably transfected IDE catalyzes a rate-limiting step in cellular insulin degradation and is localized predominantly in peroxisomes.
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PMID:Inducible expression and cellular localization of insulin-degrading enzyme in a stably transfected cell line. 807 10

Insulin-degrading enzyme (IDE), a nonlysosomal metalloprotease involved in metabolizing internalized insulin, has catalytic properties that have been strongly conserved through evolution. Two major properties distinguish IDE from the prototypic metalloprotease thermolysin. 1) It is inhibited by cysteine protease inhibitors as well as metalloprotease inhibitors; 2) it contains an inversion of the HEXXH active site motif of thermolysin, where the histidines coordinate zinc and the glutamate participates in catalysis. Furthermore, cysteine is adjacent to the glutamate residue (HXCEH) in human, rat, and Drosophila IDE, although it is not conserved in their close homologue, Escherichia coli protease III. This cysteine has been postulated to mediate the differential sensitivity of IDE and protease III to cysteine protease inhibitors and chelators. The role of the cysteine in IDE catalysis and inhibitor sensitivity was examined by mutating Cys110 to glycine or serine. To determine whether glutamate in this unusual motif participates in catalysis, we mutated Glu111 to aspartate, valine, or glutamine. Vectors containing wild type or mutant enzymes were transfected into COS cells, and expression was confirmed by Western blotting. Although the glutamate mutants were devoid of insulin degrading activity, the cysteine mutants were indistinguishable from wild type enzyme in both catalytic activity and sensitivity to inhibitors. The loss of activity in the glutamate mutants was not due to gross alterations in tertiary structure, as shown by retention of the ability to bind substrate and by conservative and nonconservative mutation of a neighboring residue with no apparent effect on catalysis. These results demonstrate that the conserved glutamate in the zinc-binding site of human insulin-degrading enzyme is a major catalytic residue, while a conserved cysteine in this region is not essential for catalysis or inhibitor sensitivity.
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PMID:Functional analysis of conserved residues in the active site of insulin-degrading enzyme. 810 41

Transtemporal approaches to the petrous apex and CP angle are standard procedures in the armamentarium of the neurotologist. In the majority of these cases, it is not possible to achieve a watertight suture closure of the dura following the procedure. Subsequently, cerebrospinal fluid leakage and potential meningitis are among the most troublesome complications for both patient and surgeon. Recent use of calcium phosphate cement (hydroxyapatite [HA]) has proved efficacious in animal studies and is now being used to close cranial defects in several medical centers, as part of an FDA-IDE study in human subjects. The use of this material is described in 11 neurotologic procedures. It is believed that hydroxyapatite cement (HAC) will become a standard tool in the management of cranial base and temporal bone defects following surgery.
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PMID:Hydroxyapatite cement: a new method for achieving watertight closure in transtemporal surgery. 810 30

Idebenone (6-(10-hydroxydecyl)-2,3-dimethoxy-5-methyl-1,4-benzoquinone) is a benzoquinone that has been shown to improve cognitive function in animals subjected to cerebral ischemia and in rats with lesions of the basal forebrain cholinergic system. Because the cognitive deficits observed in aged rats have been associated with decreased cerebral blood flow and basal forebrain cholinergic dysfunction, it was hypothesized that IDE might improve cognition in aged animals. In the present study, the effects of idebenone on cognitive function in aged Long-Evans rats were assessed using a battery of tests that evaluated attention, habituation, and spatial learning. Selective attention was assessed using an overshadowing paradigm, where IDE (30 mg/kg, IP) was injected 30 min prior to compound cue exposure. IDE enhanced the overshadowing effect in aged rats. The Morris water maze was used to assess spatial learning, where IDE (3 mg/kg, IP) was injected daily throughout the course of training. IDE did not improve the impaired performance of aged rats in the Morris task. Habituation was tested by measuring recovery from gustatory neophobia. IDE (30 mg/kg, IP) was injected 30 min prior to the first exposure to the novel taste. IDE normalized habituation rate in aged rats. It was concluded that IDE improves some forms of acquisition in aged rats, and may do so by decreasing general reactivity to novel stimuli.
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PMID:Effects of idebenone on information processing in aged Long-Evans rats. 826 97

We describe an in vitro system that will facilitate molecular analysis of the association between Lyme disease spirochetes and vector cells. We cocultured Borrelia burgdorferi continuously with two tick cell lines, RAE25 (from Rhipicephalus appendiculatus) and IDE8 (from Ixodes scapularis). A clone isolated after twenty-two passages with RAE25 cells had lost the largest (49 kb) plasmid, and probes containing information normally encoded on it, including genes for two surface proteins, hybridized to smaller plasmids. Spirochetes maintained with IDE 8 cells showed a new 43 kb plasmid that hybridized to a probe made from the 49 kb plasmid. After reisolation from hamsters, these spirochetes carried a large plasmid (100 kb) that hybridized with the 49 kb plasmid. These changes may illustrate a plasticity that enables B. burgdorferi to adapt to different environments.
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PMID:Plasmid modifications in a tick-borne pathogen, Borrelia burgdorferi, cocultured with tick cells. 826 98

Although insulin is degraded as a consequence of receptor-mediated endocytosis, the location and nature of the responsible proteinase(s) remain controversial. Insulin degrading enzyme (IDE; EC 3.4.22.11), a mainly cytosolic neutral thiol metalloendopeptidase of 110 kDa, has been proposed to be the main cellular clearance mechanism. However, endosomes concentrate and degrade internalized insulin demonstrating that IDE is unlikely to be the relevant enzyme for endosomal proteolysis of internalized insulin in liver parenchyma. In purified endosomal fractions insulin was actively degraded at acid pH and IDE was undetectable as evaluated by immunoblotting, immunoprecipitation, or chemical cross-linking procedures. Affinity purified endosomal acidic insulinase displayed a pH optimum of 4-5.5, a lack of inhibition by EDTA and N-ethylmaleimide, and a partial metal-ion requirement (for Mn2+) all of which distinguished it from IDE. A small but detectable presence of IDE in particulate nuclear (N) and large granule (ML) fractions was observed by differential centrifugation. By analytical centrifugation, IDE cosedimented with the organelle containing the peroxisomal marker proteins catalase and thiolase (median density, 1.21 g.cm-3). By preparative centrifugation, highly purified peroxisomes were observed to be enriched in IDE. Since all cloned cDNAs of IDE (human, rat, and Drosophila) reveal a deduced classical peroxisomal targeting sequence A/SKL at their carboxyl termini this may account for the peroxisomal location of IDE. Taken together, our studies identify an insulin-degrading enzyme in endosomes which is distinct from IDE. The latter's presence in peroxisomes suggests that its physiological substrate(s) in vivo are polypeptides other than insulin.
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PMID:Endosomal proteolysis of insulin by an acidic thiol metalloprotease unrelated to insulin degrading enzyme. 830 Jun 32

It has been widely accepted that in kidney, degradation of insulin occurs in lysosomes. It is thought that after internalization into the cell, insulin dissociates from its receptor, which then recycles to the plasma membrane, while the hormone is transported in endosomes to the lysosomes, where it is degraded. However, earlier studies from this laboratory have suggested that insulin may also be degraded in an extralysosomal site, most likely endosomes. Indeed, studies in other tissues, most notably liver, have shown that insulin degradation does take place in endosomes. Since the intracellular processing of insulin differs between different tissues and cell types, and as the kidney is a major site of insulin degradation, we set out to determine directly whether endosomes degrade internalized insulin in the kidney. Rats were injected with [125I]monoiodoinsulin, labeled at either the A14 or B26 tyrosine. After killing, the kidney cortex was excised, and heavy endosomes were prepared by differential and isopycnic centrifugation. The isolated [125I]insulin-loaded endosomes were incubated for up to 60 min in intracellular medium, and degradation of [125I] insulin was estimated by means of precipitation in trichloroacetic acid. In the presence of ATP (10 mM), the percent degraded was increased over the control value (no ATP present), but under these circumstances, degradation was greater when the endosomes contained internalized 125I-labeled [B26]insulin than with A14-labeled [125I]insulin (26% vs. 13% degraded/h). In the absence of ATP, the percent degraded increased when the pH of the incubation medium was lowered. Radiolabeled material was extracted from endosomes, and Sephadex G-50 analysis revealed the presence of high mol wt, insulin-size, and low mol wt material. Reverse phase HPLC analysis of the insulin-size material revealed the presence of intact insulin and a number of degradation products. The elution profiles of some of these products were consistent with that reported to arise from the action of the insulin-degrading enzyme. Western blot analysis with the antiinsulin-degrading enzyme monoclonal antibody 9B12 confirmed the presence of the enzyme in endosomal preparations. We conclude that degradation of insulin does occur in kidney cortical endosomes, probably involves the insulin-degrading enzyme, and results in the formation of relatively large intermediate products as well as low mol wt products.
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PMID:Degradation of insulin by isolated rat renal cortical endosomes. 840 92

The primary structure of the rat insulin-degrading enzyme (IDE) was determined by cDNA analysis. Rat IDE, as well as the previously characterized homologs from human and Drosophila, contain the carboxyl-terminal consensus sequence A/S-K-L, for peroxisome targeting. A stretch of 43 bp surrounding an alternatively used polyadenylation site is highly conserved between rat and human, suggesting that it may contain important regulatory information. Northern blot analysis revealed two IDE transcripts of 3.7 and 5.5 kb in various tissues. Testis was found to be exceptional in having three different RNAs (3.7, 4.1 and 6.1 kb) at a relatively high abundance. The expression of the IDE gene in testis is correlated with sexual maturation.
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PMID:The rat insulin-degrading enzyme. Molecular cloning and characterization of tissue-specific transcripts. 842 12


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