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

Abstract

Insulin-degrading enzyme (IDE) is an evolutionarily conserved ubiquitous zinc metalloprotease implicated in the efficient degradation of insulin monomer. However, IDE also degrades monomers of amyloidogenic peptides associated with disease, complicating the development of IDE inhibitors. In this work, we investigated the effects of the lipid composition of membranes on the IDE-dependent degradation of insulin. Kinetic analysis based on chromatography and insulin's helical circular dichroic signal showed that the presence of anionic lipids in membranes enhances IDE's activity toward insulin. Using NMR spectroscopy, we discovered that exchange broadening underlies the enhancement of IDE's activity. These findings, together with the adverse effects of anionic membranes in the self-assembly of IDE's amyloidogenic substrates, suggest that the lipid composition of membranes is a key determinant of IDE's ability to balance the levels of its physiologically and pathologically relevant substrates and achieve proteostasis.

Figure 1

Closed conformational state of the insulin-degrading enzyme (PDB ID: 2WBY). IDE is composed of an N-terminal half (IDE-N) and a C-terminal half (IDE-C) joined together by an unstructured linker (magenta). When in its closed conformation, IDE forms a catalytic chamber that can only accommodate small monomeric substrates such as insulin and Aβ42. IDE-N contains a conserved exosite (yellow), which has been hypothesized to anchor the substrate prior to degradation. IDE has the HXXEH motif, which contains the two histidine residues (H108 and H112) that coordinate Zn²⁺ and the catalytically important glutamate residue (E111). The arrow in the lower half of IDE-N indicates the relative location of the catalytic Zn²⁺ ion.

Figure 2

IDE-dependent degradation of insulin in the absence or presence of SUVs follows Michaelis–Menten kinetics. Plots of V₀ against insulin concentration are hyperbolic. Each data point represents the mean from three kinetic trials, and the error bars are standard deviations. The lines are fits to the Michaelis–Menten equation.

Figure 3

IDE-dependent degradation of insulin monitored by NMR. One-dimensional ¹H NMR spectra of insulin digestion reactions in (A) the absence of SUVs, and the presence of SUVs composed of (B) 100% DOPC, (C) DOPC/DOPS (7:3 mol/mol), and (D) 100% DOPS. In the absence of IDE, the broadening of the peaks in the aromatic region (6.6–7.4 ppm) in samples that contain membranes with anionic DOPS increased. In the presence of IDE, the broad peaks in the aromatic region are replaced by sharp peaks due to insulin fragments that tumble rapidly in solution. All spectra were recorded at 37 °C. The NMR samples were incubated at 37 °C in between the acquisition of spectra. The concentration of insulin in all samples was set at 100 μM. At this concentration, insulin exists mainly as hexamers. The insulin-to-lipid and the substrate-to-enzyme molar ratios were set at 1:50 and 100:1, respectively.

Figure 4

Chemical exchange in insulin. Oligomeric insulin and insulin monomer are in dynamic equilibrium with one another. This equilibrium is characterized by the exchange rate k_ex. The distribution of oligomers is indicated by n, which ranges from 2 to 6. At the concentration of insulin used in the NMR studies, hexamers are the dominant oligomers.