Somatostatin: A Novel Substrate and a Modulator of Insulin-Degrading Enzyme Activity

Abstract

Insulin-degrading enzyme (IDE) is an interesting pharmacological target for Alzheimer's disease (AD), since it hydrolyzes β-amyloid, producing nonneurotoxic fragments. It has also been shown that the somatostatin level reduction is a pathological feature of AD and that it regulates the neprilysin activity toward β-amyloid.In this work, we report for the first time that IDE is able to hydrolyze somatostatin [k cat (s − 1 ) = 0.38 (±0.05); K m (M) = 7.5 (±0.9)× 10 − 6 ] at the Phe6-Phe7 amino acid bond. On the other hand, somatostatin modulates IDE activity, enhancing the enzymatic cleavage of a novel fluorogenic β-amyloid through a decrease of the K m toward this substrate, which corresponds to the 10-25 amino acid sequence of the Aβ(1-40). Circular dichroism spectroscopy and surface plasmon resonance imaging experiments show that somatostatin binding to IDE brings about a concentration-dependent structural change of the secondary and tertiary structure(s) of the enzyme, revealing two possible binding sites. The higher affinity binding site disappears upon inactivation of IDE by ethylenediaminetetraacetic acid, which chelates the catalytic Zn 2+ ion. As a whole, these features suggest that the modulatory effect is due to an allosteric mechanism: somatostatin binding to the active site of one IDE subunit (where somatostatin is cleaved) induces an enhancement of IDE proteolytic activity toward fluorogenic β-amyloid by another subunit. Therefore, this investigation on IDE-somatostatin interaction contributes to a more exhaustive knowledge about the functional and structural aspects of IDE and its pathophysiological implications in the amyloid deposition and somatostatin homeostasis in the brain.

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.…”

“…Since the discovery of insulin and Aβ cleavage by IDE [11], much effort has been put in trying to understand some key questions regarding cleavage sites [12,13], kinetics of substrate interaction [14,15] and the mechanism of IDE modulation [16,17].…”