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Purified recombinant insulin-degrading enzyme degrades amyloid beta-protein but does not promote its oligomerization.

ABSTRACT: 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.

SUBMITTER: Chesneau V

PROVIDER: S-EPMC1221388 | biostudies-other | 2000 Oct

REPOSITORIES: biostudies-other

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Project description:BACKGROUND:Alzheimer's disease (AD) is characterized by the deposition of beta-amyloid (Abeta) peptides in the brain extracellular matrix, resulting in pathological changes and neurobehavioral deficits. Previous work from this laboratory demonstrated that the choroid plexus (CP) possesses the capacity to remove Abeta from the cerebrospinal fluid (CSF), and exposure to lead (Pb) compromises this function. Since metalloendopeptidase insulin-degrading enzyme (IDE), has been implicated in the metabolism of Abeta, we sought to investigate whether accumulation of Abeta following Pb exposure was due to the effect of Pb on IDE. METHODS:Rats were injected with a single dose of Pb acetate or an equivalent concentration of Na-acetate; CP tissues were processed to detect the location of IDE by immunohistochemistry. For in vitro studies, choroidal epithelial Z310 cells were treated with Pb for 24 h in the presence or absence of a known IDE inhibitor, N-ethylmaleimide (NEM) to assess IDE enzymatic activity and subsequent metabolic clearance of Abeta. Additionally, the expression of IDE mRNA and protein were determined using real time PCR and western blots respectively. RESULTS:Immunohistochemistry and confocal imaging revealed the presence of IDE towards the apical surface of the CP tissue with no visible alteration in either its intensity or location following Pb exposure. There was no significant difference in the expressions of either IDE mRNA or protein following Pb exposure compared to controls either in CP tissues or in Z310 cells. However, our findings revealed a significant decrease in the IDE activity following Pb exposure; this inhibition was similar to that seen in the cells treated with NEM alone. Interestingly, treatment with Pb or NEM alone significantly increased the levels of intracellular Abeta, and a greater accumulation of Abeta was seen when the cells were exposed to a combination of both. CONCLUSION:These data suggest that Pb exposure inhibits IDE activity but does not affect its expression in the CP. This, in turn, leads to a disrupted metabolism of Abeta resulting in its accumulation at the blood-CSF barrier.

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Purified recombinant insulin-degrading enzyme degrades amyloid beta-protein but does not promote its oligomerization.

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