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.

Modulation of the Activity of the Insulin-Degrading Enzyme by Aβ Peptides

“…As such, the initial velocity (V 0 ) of insulin proteolysis can be determined from [θ obs(222 nm) ] recorded within the first few minutes of the proteolysis, as previously described. 34,36,37 Unaggregated Aβ (1−40) or Aβ(pyroE3−42) is unstructured, 38,39 and thus, the decrease in [θ obs (222 nm) ] in samples that contain insulin and Aβ peptide must be due to insulin degradation. Figure 3 presents the dependence of V 0 on substrate concentration for the IDE-dependent degradation of insulin in the absence of Aβ peptides (control), in the presence of 5 μM Aβ(1−40), and in the presence of 5 μM Aβ(pyroE3− 42).…”

“…The Michaelis–Menten kinetic constants for the IDE-dependent degradation of insulin in the absence and presence of Aβ(1–40) or Aβ­(pyroE3–42) can be determined using CD spectroscopy. − The basic premise of this approach is simple: the α-helical dichroic spectrum of insulin is sensitive to the extent of proteolytic degradation by IDE. As noted elsewhere, ,, the magnitude of insulin’s ellipticity at 222 nm (i.e., ([θ obs (222 nm) ]) decreases as digestion proceeds.…”

“…The Michaelis–Menten kinetic constants for the IDE-dependent degradation of insulin in the absence and presence of Aβ(1–40) or Aβ­(pyroE3–42) can be determined using CD spectroscopy. − The basic premise of this approach is simple: the α-helical dichroic spectrum of insulin is sensitive to the extent of proteolytic degradation by IDE. As noted elsewhere, ,, the magnitude of insulin’s ellipticity at 222 nm (i.e., ([θ obs (222 nm) ]) decreases as digestion proceeds. As such, the initial velocity ( V 0 ) of insulin proteolysis can be determined from [θ obs (222 nm) ] recorded within the first few minutes of the proteolysis, as previously described. ,, Unaggregated Aβ(1–40) or Aβ­(pyroE3–42) is unstructured, , and thus, the decrease in [θ obs (222 nm) ] in samples that contain insulin and Aβ peptide must be due to insulin degradation.…”

“…To remove preformed aggregates, Aβ(1–40) and Aβ­(pyroE3–42) were pretreated and lyophilized as previously described and stored at −20 °C until ready for use. All protein solutions were prepared fresh in 50 mM Tris (pH 7.4), and their concentrations were determined by UV absorbance using molar extinction coefficients at 275 nm of 6190 M –1 cm –1 and 1390 M –1 cm –1 for insulin and Aβ, respectively. ,, …”

“…Michaelis–Menten kinetic parameters for the IDE-dependent degradation of insulin in the absence or presence of Aβ(1–40) or Aβ­(pyroE3–42) were determined using the CD-based kinetic assay that we have previously reported. ,, Briefly, insulin in 50 mM Tris buffer (pH 7.4) with concentrations of 15–110 μM was prepared in the absence of Aβ (control) or in the presence of 5 μM Aβ(1–40) or 5 μM Aβ­(pyroE3–42). Proteolysis in each sample was initiated with the addition of 1 μM IDE.…”