Insulin-Degrading Enzyme: a possible “dead-end” chaperone for amyloid peptides

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

Chicago

Congreso; 11th International Conference on Alzheimer's Disease; 2008

Alzheimer`s Disease Association

Insulin-degrading enzyme (IDE) is central to the turnover of insulin and degrades amyloid beta(Ab) 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 Ab 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. Ab sequence 17-27, known to be crucial in amyloid assembly was sufficient to form a stable complex with IDE. Monomeric as opposed to aggregated Ab 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 Ab takes place with at least part of the substrate binding site of the protease. Limited proteolysis showed that Ab remained bound to a ~25 kDa Nterminal fragment of IDE in an SDS-resistant manner. Mass spectrometry after in gel digestion of the IDE-Ab complex showed that peptides derived from the region that includes the catalytic site of IDE were recovered with Ab. Taken together, these results are suggestive of an unprecedented mechanism of 1 is central to the turnover of insulin and degrades amyloid beta (Ab) 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 Ab that resisted dissociation after treatment with strong denaturants. This interaction was also observed with rat brain IDE and detected in a SDS-soluble fraction from AD cortical tissue. Ab sequence 17-27, known to be crucial in amyloid assembly was sufficient to form a stable complex with IDE. Monomeric as opposed to aggregated Ab 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 Ab takes place with at least part of the substrate binding site of the protease. Limited proteolysis showed that Ab remained bound to a ~25 kDa Nterminal fragment of IDE in an SDS-resistant manner. Mass spectrometry after in gel digestion of the IDE-Ab complex showed that peptides derived from the region that includes the catalytic site of IDE were recovered with Ab. Taken together, these results are suggestive of an unprecedented mechanism of conformation-dependent substrate binding that may perturb Ab clearance, insulin turnover and promote AD pathogenesis.