Neurons regulate extracellular levels of amyloid beta-protein via proteolysis by insulin-degrading enzyme.
Progressive cerebral accumulation of amyloid beta-protein (Abeta) is an early and invariant feature of Alzheimer's disease. Little is known about how Abeta, after being secreted, is degraded and cleared from the extracellular space of the brain. Defective Abeta degradation could be a risk factor for the development of Alzheimer's disease in some subjects. We reported previously that microglial cells release substantial amounts of an Abeta-degrading protease that, after purification, is indistinguishable from insulin-degrading enzyme (IDE). Here we searched for and characterized a role for IDE in Abeta degradation by neurons, the principal cell type that produces Abeta. Whole cultures of differentiated pheochromocytoma (PC12) cells and primary rat cortical neurons actively degraded endogenously secreted Abeta via IDE. However, unlike that in microglia, IDE in differentiated neurons was not released but localized to the cell surface, as demonstrated by biotinylation. Undifferentiated PC12 cells released IDE into their medium, whereas after differentiation, IDE was cell associated but still degraded Abeta in the medium. Overexpression of IDE in mammalian cells markedly reduced the steady-state levels of extracellular Abeta(40) and Abeta(42), and the catalytic site mutation (E111Q) abolished this effect. We observed a novel membrane-associated form of IDE that is approximately 5 kDa larger than the known cytosolic form in a variety of cells, including differentiated PC12 cells. Our results support a principal role for membrane-associated and secreted IDE isoforms in the degradation and clearance of naturally secreted Abeta by neurons and microglia. (+info)
Degradation of amylin by insulin-degrading enzyme.
A pathological feature of Type 2 diabetes is deposits in the pancreatic islets primarily composed of amylin (islet amyloid polypeptide). Although much attention has been paid to the expression and secretion of amylin, little is known about the enzymes involved in amylin turnover. Recent reports suggest that insulin-degrading enzyme (IDE) may have specificity for amyloidogenic proteins, and therefore we sought to determine whether amylin is an IDE substrate. Amylin-degrading activity co-purified with IDE from rat muscle through several chromatographic steps. Metalloproteinase inhibitors inactivated amylin-degrading activity with a pattern consistent with the enzymatic properties of IDE, whereas inhibitors of acid and serine proteases, calpains, and the proteasome were ineffective. Amylin degradation was inhibited by insulin in a dose-dependent manner, whereas insulin degradation was inhibited by amylin. Other substrates of IDE such as atrial natriuretic peptide and glucagon also competitively inhibited amylin degradation. Radiolabeled amylin and insulin were both covalently cross-linked to a protein of 110 kDa, and the binding was competitively inhibited by either unlabeled insulin or amylin. Finally, a monoclonal anti-IDE antibody immunoprecipitated both insulin- and amylin-degrading activities. The data strongly suggest that IDE is an amylin-degrading enzyme and plays an important role in the clearance of amylin and the prevention of islet amyloid formation. (+info)
Purified recombinant insulin-degrading enzyme degrades amyloid beta-protein but does not promote its oligomerization.
Amyloid beta-protein (Abeta) has been implicated as an early and essential factor in the pathogenesis of Alzheimer's disease. Although its cellular production has been studied extensively, little is known about Abeta clearance. Recently, insulin-degrading enzyme (IDE), a 110-kDa metalloendopeptidase, was found to degrade both endogenously secreted and synthetic Abeta peptides. Surprisingly, IDE-mediated proteolysis of [(125)I]Abeta(1-40) in microglial cell-culture media was accompanied by the formation of (125)I-labelled peptides with higher apparent molecular masses, raising the possibility that the degradation products act as 'seeds' for Abeta oligomerization. To directly address the role of IDE in Abeta degradation and oligomerization, we investigated the action of purified recombinant wild-type and catalytically inactive IDEs. Our data demonstrate that (i) IDE alone is sufficient to cleave purified Abeta that is either unlabelled, iodinated or (35)S-labelled; (ii) the initial cleavage sites are His(14)-Gln(15), Phe(19)-Phe(20) and Phe(20)-Ala(21); and (iii) incubation of IDE with [(125)I]Abeta, but not with [(35)S]-Abeta, leads to the formation of slower migrating species on gels. Since iodination labels N-terminal fragments of Abeta, and (35)S labels C-terminal products, we analysed unlabelled synthetic fragments of Abeta and determined that only the N-terminal fragments migrate with anomalously high molecular mass. These results indicate that IDE alone is sufficient to degrade Abeta at specific sites, and that its degradation products do not promote oligomerization of the intact Abeta peptide. (+info)
Analysis of the subsite specificity of rat insulysin using fluorogenic peptide substrates.
Recombinant rat insulysin was shown to cleave the internally quenched fluorogenic peptide 2-aminobenzyl-GGFLRKVGQ-ethylenediamine-2,4-dinitrophenol at the R-K bond, exhibiting a K(m) of 13 microm and a V(max) of 2.6 micromol min(-1) mg(-1). Derivatives of this peptide in which the P(2) leucine or the P(2)' valine were replaced with other residues were used to probe the subsite specificity of the enzyme. Varying the P(2) residue produced a 4-fold range in K(m) and a 7-fold range in k(cat). The nature of the P(2) residue had a significant effect on the site of cleavage. Leucine, isoleucine, valine, and aspartate produced cleavage at the R-K bond. Asparagine produced 36% cleavage at the N-R bond and 64% cleavage at the R-K bond, whereas with alanine or serine the A-R and S-R bonds were the major cleavage sites. With tyrosine, phenylalanine, methionine, or histidine representing the varied residue X, cleavages at F-X, X-R, and R-K were seen, whereas with tryptophan equal cleavage occurred at the F-W and W-R bonds. Variable P(2)' residues produce less of a change in both K(m) and k(cat) and have little influence on the cleavage site. Exceptions are phenylalanine, tyrosine, leucine, and isoleucine, which in addition to producing cleavage at the R-K bond, produce significant cleavage at the L-R bond. Alanine and tyrosine were unique in producing cleavage at the F-L bond. Taken together, these data suggest that insulysin specificity is directed toward the amino side of hydrophobic and basic residues and that the enzyme has an extended substrate binding site. (+info)
Insulysin hydrolyzes amyloid beta peptides to products that are neither neurotoxic nor deposit on amyloid plaques.
Insulysin (EC. 18.104.22.168) has been implicated in the clearance of beta amyloid peptides through hydrolytic cleavage. To further study the action of insulysin on Abeta peptides recombinant rat insulysin was used. Cleavage of both Abeta(1-40) and Abeta(1-42) by the recombinant enzyme was shown to initially occur at the His(13)-His(14), His(14)-Gln(15), and Phe(19)-Phe(20) bonds. This was followed by a slower cleavage at the Lys(28)-Gly(29), Val(18)-Phe(19), and Phe(20)-Ala(21) positions. None of the products appeared to be further metabolized by insulysin. Using a rat cortical cell system, the action of insulysin on Abeta(1-40) and Abeta(1-42) was shown to eliminate the neurotoxic effects of these peptides. Insulysin was further shown to prevent the deposition of Abeta(1-40) onto a synthetic amyloid. Taken together these results suggest that the use of insulysin to hydrolyze Abeta peptides represents an alternative gene therapeutic approach to the treatment of Alzheimer's disease. (+info)
Evidence for genetic linkage of Alzheimer's disease to chromosome 10q.
Recent studies suggest that insulin-degrading enzyme (IDE) in neurons and microglia degrades Abeta, the principal component of beta-amyloid and one of the neuropathological hallmarks of Alzheimer's disease (AD). We performed parametric and nonparametric linkage analyses of seven genetic markers on chromosome 10q, six of which map near the IDE gene, in 435 multiplex AD families. These analyses revealed significant evidence of linkage for adjacent markers (D10S1671, D10S583, D10S1710, and D10S566), which was most pronounced in late-onset families. Furthermore, we found evidence for allele-specific association between the putative disease locus and marker D10S583, which has recently been located within 195 kilobases of the IDE gene. (+info)
Insulin-degrading enzyme exists inside of rat liver peroxisomes and degrades oxidized proteins.
Insulin-degrading enzyme (IDE) was detected by immunoblot analysis in highly purified rat liver peroxisomes. IDE in the peroxisomal fraction was resistant to proteolysis by trypsin and chymotrypsin under conditions where the peroxisomal membranes remained intact. After sonication of the peroxisomal fraction, IDE was recovered in the supernatant fraction. Further, the localization of IDE in the peroxisomes was shown by immunoelectron microscopy. In addition, IDE isolated from peroxisomes degraded insulin as well as oxidized lysozyme as a model substrate for oxidized proteins. These results suggest that IDE exists in an active form in the matrix of rat liver peroxisomes and is involved in elimination of oxidized proteins in peroxisomes. (+info)
Stimulation of beta-amyloid precursor protein trafficking by insulin reduces intraneuronal beta-amyloid and requires mitogen-activated protein kinase signaling.
Alzheimer's Disease (AD) is characterized by cerebral accumulation of beta-amyloid peptides (Abeta), which are proteolytically derived from beta-amyloid precursor protein (betaAPP). betaAPP metabolism is highly regulated via various signal transduction systems, e.g., several serine/threonine kinases and phosphatases. Several growth factors known to act via receptor tyrosine kinases also have been demonstrated to regulate sbetaAPP secretion. Among these receptors, insulin and insulin-like growth factor-1 receptors are highly expressed in brain, especially in hippocampus and cortex. Emerging evidence indicates that insulin has important functions in brain regions involved in learning and memory. Here we present evidence that insulin significantly reduces intracellular accumulation of Abeta and that it does so by accelerating betaAPP/Abeta trafficking from the trans-Golgi network, a major cellular site for Abeta generation, to the plasma membrane. Furthermore, insulin increases the extracellular level of Abeta both by promoting its secretion and by inhibiting its degradation via insulin-degrading enzyme. The action of insulin on betaAPP metabolism is mediated via a receptor tyrosine kinase/mitogen-activated protein (MAP) kinase kinase pathway. The results suggest cell biological and signal transduction mechanisms by which insulin modulates betaAPP and Abeta trafficking in neuronal cultures. (+info)