Insulin-degrading enzyme: a possible “dead-end” chaperone for amyloid peptides.

DI TULLIO MATIAS; FERNANDEZ-GAMBA AGATA; LLOVERA RAMIRO; MORELLI LAURA; CASTAÑO EDUARDO

Chicago, USA.

Congreso; International conferences of Alzheimer`s disease and related disorders; 2008

Alzheimer`s Disease Association

Insulin-degrading enzyme (IDE)1 is central to the turnover of insulin and degrades amyloid â (Aâ) in the mammalian brain. Biochemical and genetic data support that IDE may play a role in late onset Alzheimer’s disease (AD) and recent studies suggest an association between AD and diabetes mellitus type 2 (DM2). Here we show that a natively folded recombinant IDE was capable of forming a stable complex with Aâ that resisted dissociation after treatment with strong denaturants. This interaction was alsoobserved with rat brain IDE and detected in a SDS-soluble fraction from AD cortical tissue. Aâ sequence 17-27, known to be crucial in amyloid assembly was sufficient to form a stable complex with IDE. Monomeric as opposed to aggregated Aâ was competent to associate irreversibly with IDE following a very slow kinetics (t1/2 ~45 min). Partial denaturation of IDE as well as pre-incubation with a 10-fold molar excess of insulin prevented complex formation, suggesting that the irreversible interaction of Aâ takes place with at least part of the substrate binding site of the protease. Limited proteolysis showed that Aâ remained bound to a ~25 kDa Nterminal fragment of IDE in an SDS-resistant manner. Mass spectrometry after in gel digestion of the IDE-Aâ complex showed that peptides derived from the region that includes the catalytic site of IDE were recovered with Aâ. Taken together, these results are suggestive of an unprecedented mechanism of 1 is central to the turnover of insulin and degrades amyloid â (Aâ) in the mammalian brain. Biochemical and genetic data support that IDE may play a role in late onset Alzheimer’s disease (AD) and recent studies suggest an association between AD and diabetes mellitus type 2 (DM2). Here we show that a natively folded recombinant IDE was capable of forming a stable complex with Aâ that resisted dissociation after treatment with strong denaturants. This interaction was alsoobserved with rat brain IDE and detected in a SDS-soluble fraction from AD cortical tissue. Aâ sequence 17-27, known to be crucial in amyloid assembly was sufficient to form a stable complex with IDE. Monomeric as opposed to aggregated Aâ was competent to associate irreversibly with IDE following a very slow kinetics (t1/2 ~45 min). Partial denaturation of IDE as well as pre-incubation with a 10-fold molar excess of insulin prevented complex formation, suggesting that the irreversible interaction of Aâ takes place with at least part of the substrate binding site of the protease. Limited proteolysis showed that Aâ remained bound to a ~25 kDa Nterminal fragment of IDE in an SDS-resistant manner. Mass spectrometry after in gel digestion of the IDE-Aâ complex showed that peptides derived from the region that includes the catalytic site of IDE were recovered with Aâ. Taken together, these results are suggestive of an unprecedented mechanism of conformation-dependent substrate binding that may perturb Aβ clearance, insulin turnover and promote AD pathogenesis.