Differential Degradation of Amyloid β Genetic Variants Associated with Hereditary Dementia or Stroke by Insulin-degrading Enzyme

Abstract

Inherited amino acid substitutions at position 21, 22, or 23 of amyloid ␤ (A␤) lead to presenile dementia or stroke. Insulin-degrading enzyme (IDE) can hydrolyze A␤ wild type, yet whether IDE is capable of degrading A␤ bearing pathogenic substitutions is not known. We studied the degradation of all of the published A␤ genetic variants by recombinant rat IDE (rIDE). Monomeric A␤ wild type, Flemish (A21G), Italian (E22K), and Iowa (D23N) variants were readily degraded by rIDE with a similar efficiency. However, proteolysis of A␤ Dutch (E22Q) and Arctic (E22G) was significantly lower as compared with A␤ wild type and the rest of the mutant peptides. In the case of A␤ Dutch, inefficient proteolysis was related to a high content of ␤ structure as assessed by circular dichroism. All of the A␤ variants were cleaved at Glu 3 -Phe 4 and Phe 4 -Arg 5 in addition to the previously described major sites within positions 13-15 and 18 -21. SDS-stable A␤ dimers were highly resistant to proteolysis by rIDE regardless of the variant, suggesting that IDE recognizes a conformation that is available for interaction only in monomeric A␤. These results raise the possibility that upregulation of IDE may promote the clearance of soluble A␤ in hereditary forms of A␤ diseases.The accumulation of amyloid ␤ peptide (A␤) 1 in the brain is a central process in a number of human neurodegenerative disorders that may be grouped as "amyloid ␤ diseases" (1). In Alzheimer's disease, A␤ is mainly found within senile plaques in the neurophil and vascular lesions, whereas in sporadic and hereditary amyloid angiopathies, A␤ deposits are mainly associated with cortical and leptomeningeal vessels leading to stroke or multi-infarct dementia. To a lesser extent, cerebral A␤ deposits are also present in normal aging (2). Autosomal dominant mutations in the amyloid ␤ precursor protein (A␤ PP) gene result in amino acid substitutions at position 21, 22, or 23 of A␤ sequence. Although these A␤ variants present with a primarily vascular deposition, they translate into different clinical phenotypes. In this regard, A␤ Arctic (E22G) and A␤ Iowa (D23N) are characterized by presenile dementia and A␤ Flemish (A21G) is associated with early onset dementia and cerebral hemorrhage, whereas A␤ Dutch (E22Q) and A␤ Italian (E22K) variants have a predominant vascular phenotype characterized by massive strokes (3-7). The underlying mechanism of aggregation and deposition in vivo may be strongly influenced by the type of amino acid substitution as well as the location of mutations in the A␤ peptide. In vitro studies have shown that A␤ E22Q and A␤ D23N form typical amyloid fibrils at a higher rate than wild-type A␤ and that A␤ E22G assembles into unique protofibrils that may be toxic to neurons (3, 8 -10). In the case of A␤ A21G, overproduction of the peptide may contribute as a pathogenic mechanism (11, 12). Regarding A␤ E22K, deposition seems not to be related with fibril formation rate, and yet this variant may be toxic to human cerebrovascular smooth muscle cells in cult...

Metal ions affect insulin-degrading enzyme activity

“…This inhibition is even more significant in the case of silver(I) (which is employed instead of copper(I), due to the higher stability of the monovalent form under the experimental conditions), such that the enzyme is completely inactive toward the B20-30 peptide, similar to what has been observed in the case of the Aβ [16][17][18][19][20][21][22][23][24][25][26][27][28] peptide [54], and it is not reported in Fig. 6.…”

“…A very important aspect to be remarked is the fact that K Cu (=6.3(±2.1) × 10 − 6 M, see Table 3), resulting from the application of Scheme 1, is drastically different from the value reported before for the copper(II) effect on the enzymatic processing of Aβ [16][17][18][19][20][21][22][23][24][25][26][27][28] [54]. Since K Cu referred to the interaction of copper(II) with free IDE, its value must be independent on the substrate.…”

“…It is also interesting to outline that if we compare these parameters with those obtained for the amyloid peptides Aβ [1][2][3][4][5][6][7][8][9][10][11][12][13][14][15][16] and Aβ [16][17][18][19][20][21][22][23][24][25][26][27][28] several similarities and some differences emerge [54]. In particular, the overall proteolytic activity toward the main cleavage sites (i.e., Phe 24 [16][17][18][19][20][21][22][23][24][25][26][27][28] ) is somewhat higher in the case of the B20-30 peptide due to a slightly more efficient cleavage rate-limiting step (see Table 2), whereas the substrate affinity of B20-30 appears intermediate between that of Aβ [1][2][3]…”

“…This hypothesis is clearly incompatible with the copper(II) effect on the enzymatic processing of the peptide B20-30 by IDE, forcing us to employ a value of β ≠ 0. A new analysis of the copper(II) effect on the cleavage of Aβ [16][17][18][19][20][21][22][23][24][25][26][27][28] by IDE resulted in the following parameters (i.e., K Cu = 6.3(±2.1) × 10 − 6 M, α = 0.5 ± 0.2 and β = 0.32 ± 0.05), where the value of K Cu was clearly imposed and the obtained α and β parameters can be compared with those obtained for the processing of B20-30.…”

“…It is often reported that the common feature shared by IDE substrates is the ability to form, under certain physiological conditions, amyloid fibrils [18][19][20][21]. Furthermore, several evidences indicate that the selective binding of IDE's substrates and their unfolding and processing are driven by the unique size, shape and electrostatic potential of the catalytic chamber and by interaction with the exosite [22,23].…”