Quantitative NMR Study of Insulin-Degrading Enzyme Using Amyloid-β and HIV-1 p6 Elucidates Its Chaperone Activity

Abstract

Insulin-degrading enzyme (IDE) hydrolyzes monomeric polypeptides, including amyloid-β (Aβ) and HIV-1 p6. It also acts as a nonproteolytic chaperone to prevent Aβ polymerization. Here we compare interactions of Aβ and non-amyloidogenic p6 with IDE. Although both exhibited similar proteolysis rates, the binding kinetics to an inactive IDE characterized using relaxation-based NMR were remarkably different. IDE and Aβ formed a sparsely populated complex with a lifetime of milliseconds in which a short hydrophobic cleavage segment of Aβ was anchored to IDE. Strikingly, a second and more stable complex was significantly populated with a subsecond lifetime owing to multiple intermolecular contacts between Aβ and IDE. By selectively sequestering Aβ in this nonproductive complex, IDE likely increases the critical concentration required for fibrillization. In contrast, IDE and p6 formed a transient, submillisecond complex involving a single anchoring p6 motif. Modulation of intermolecular interactions, thus, allows IDE to differentiate between non-amyloidogenic and amyloidogenic substrates.

Exchange Broadening Underlies the Enhancement of IDE-Dependent Degradation of Insulin by Anionic Membranes

“…This conclusion is also supported by 2D NOESY NMR of insulin in the absence of IDE (Figure S5), which shows the absence of cross peaks due to insulin, in sharp contrast to NOESY spectra we reported for insulin at pH 2 in the absence of SUVs . Additionally, our NOESY data (Figure S5) indicate that multidimensional relaxation-based NMR methods that were recently used to investigate the interaction of Aβ42 with IDE do not apply . In the presence of SUVs containing anionic lipids and in the absence of IDE, the broadening of the peaks in the aromatic regions of the spectra shown in Figure C,D increased.…”

Mechanistic roles of tyrosine phosphorylation in reversible amyloids, autoinhibition, and endosomal membrane association of ALIX

“…The purification protocol for TEV protease is described in our previous works ( 16 , 51 ). Briefly, the cells were resuspended in a lysis buffer containing 50 mM Tris, pH 8.0, and 250 mM NaCl.…”

“…All protein constructs, the end products of in vitro phosphorylation, and site-specific spin labeling reactions (see below) were verified by MS using our previously published protocols ( 16 , 51 ). Briefly, an Agilent 6230 TOF-mass spectrometer with Jet Stream ESI was used for LC–ESI–TOFMS analysis.…”

Insulin-Degrading Enzyme, an Under-Estimated Potential Target to Treat Cancer?

“…The involvement of IDE in the degradation of all these substrates suggests its potential role in processes modulated by these peptides. Interestingly, most substrates of IDE share a propensity to be amyloidogenic peptides [ 63 ], with a few exceptions like HIV-1 p6 [ 64 ].…”

“…Interestingly, α-synuclein has a dual effect in cancer by promoting tumorigenicity and inhibiting cancer growth according to the models studied in the different studies [ 72 ]. Ramaraju et al describe the kinetic model of two IDE-bound states, one used for proteolysis and the other as a kinetic trap to differentiate non-amyloidogenic and amyloidogenic substrates [ 64 ]. Finally, IDE interacts with intermediate filaments, vimentin and nestin complexes during mitosis.…”