The catalytic domain of insulin-degrading enzyme forms a denaturant-resistant complex with amyloid beta peptide: implications for Alzheimer disease pathogenesis.
Insulin-degrading enzyme (IDE) is central to the turnover of insulin and degrades amyloid beta (Abeta) in the mammalian brain. Biochemical and genetic data support the notion that IDE may play a role in late onset Alzheimer disease (AD), and recent studies suggest an association between AD and diabetes mellitus type 2. Here we show that a natively folded recombinant IDE was capable of forming a stable complex with Abeta that resisted dissociation after treatment with strong denaturants. This interaction was also observed with rat brain IDE and detected in an SDS-soluble fraction from AD cortical tissue. Abeta sequence 17-27, known to be crucial in amyloid assembly, was sufficient to form a stable complex with IDE. Monomeric as opposed to aggregated Abeta was competent to associate irreversibly with IDE following a very slow kinetics (t(1/2) approximately 45 min). Partial denaturation of IDE as well as preincubation with a 10-fold molar excess of insulin prevented complex formation, suggesting that the irreversible interaction of Abeta takes place with at least part of the substrate binding site of the protease. Limited proteolysis showed that Abeta remained bound to a approximately 25-kDa N-terminal fragment of IDE in an SDS-resistant manner. Mass spectrometry after in gel digestion of the IDE .Abeta complex showed that peptides derived from the region that includes the catalytic site of IDE were recovered with Abeta. Taken together, these results are suggestive of an unprecedented mechanism of conformation-dependent substrate binding that may perturb Abeta clearance, insulin turnover, and promote AD pathogenesis.
Llovera
Ramiro E
RE
Fundación Instituto Leloir-Instituto de Investigaciones Bioquímicas de Buenos Aires, Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET), 435 Av. Patricias Argentinas, Ciudad de Buenos Aires C1405BWE, Argentina.
de Tullio
Matías
M
Alonso
Leonardo G
LG
Leissring
Malcolm A
MA
Kaufman
Sergio B
SB
Roher
Alex E
AE
de Prat Gay
Gonzalo
G
Morelli
Laura
L
Castaño
Eduardo M
EM
D000544:Alzheimer Disease / Q000378:metabolism / Q000473:pathology*; D016229:Amyloid beta-Peptides / Q000737:chemistry*; D000818:Animals; D001665:Binding Sites; D001921:Brain / Q000378:metabolism; D020134:Catalytic Domain; D006801:Humans; D007339:Insulysin / Q000737:chemistry*; D007700:Kinetics; D013058:Mass Spectrometry; D008954:Models, Biological; D011485:Protein Binding; D051381:Rats; D012542:Scattering, Radiation; D013379:Substrate Specificity
D016229:Amyloid beta-Peptides; D007339:Insulysin