Role of the Metal Center in the Modulation of the Aggregation Process of Amyloid Model Systems by Square Planar Complexes Bearing 2-(2'-pyridyl)benzimidazole Ligands

The effect of analogue Pd(II)-, Pt(II)-, and Au(III) compounds featuring 2-(2'-pyridyl)benzimidazole on the aggregation propensity of amyloid-like peptides derived from Aβ and from the C-terminal domain of nucleophosmin 1 was investigated. Kinetic profiles of aggregation were evaluated using thioflavin binding assays, whereas the interactions of the compounds with the peptides were studied by UV-Vis absorption spectroscopy and electrospray ionization mass spectrometry. The results indicate that the compounds modulate the aggregation of the investigated peptides using different mechanisms, suggesting that the reactivity of the metal center and the physicochemical properties of the metals (rather than those of the ligands and the geometry of the metal compounds) play a crucial role in determining the anti-aggregation properties

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

Insulin-degrading enzyme (IDE) is an interesting pharmacological target for Alzheimer's disease (AD), since it hydrolyzes beta-amyloid, producing non-neurotoxic 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 beta-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) x 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 beta-amyloid through a decrease of the K-m toward this substrate, which corresponds to the 10-25 amino acid sequence of the A beta(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 ethylenediaminetetra acetic acid, which chelates, the catalytic Zn2+ 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 beta-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. (C) 2008 Elsevier Ltd. All rights reserved

Ruthenium complexes bearing glucosyl ligands are able to inhibit the amyloid aggregation of short Histidine-peptides

Neurodegenerative diseases are often characterized by the formation of aggregates of amyloidogenic peptides and proteins, facilitating the formation of neurofibrillary plaques. In this study, we investigate a series of Ru-complexes sharing three-legged piano-stool structures based on the arene ring and glucosylated carbene ligands. The ability of these complexes to bind amyloid His-peptides was evaluated by ESI-MS, and their effects on the aggregation process were investigated through ThT and Tyr fluorescence emission. The complexes were demonstrated to bind the amyloidogenic peptides even with different mechanisms and kinetics depending on the chemical nature of the ligands around the Ru(II) ion. TEM analysis detected the disaggregation of typical fibers caused by the presence of Ru-compounds. Overall, our results show that the Ru-complexes can modulate the aggregation of His-amyloids and can be conceived as good lead compounds in the field of novel anti-aggregating agents in neurodegeneration

Inhibition of Apurinic/apyrimidinic endonuclease I’s redox activity revisited

The essential base excision repair protein, apurinic/apyrimidinic endonuclease 1 (APE1), plays an important role in redox regulation in cells and is currently targeted for the development of cancer therapeutics. One compound that binds APE1 directly is (E)-3-[2-(5,6-dimethoxy-3-methyl-1,4-benzoquinonyl)]-2-nonylpropenoic acid (E3330). Here, we revisit the mechanism by which this negatively charged compound interacts with APE1 and inhibits its redox activity. At high concentrations (millimolar), E3330 interacts with two regions in the endonuclease active site of APE1, as mapped by hydrogen–deuterium exchange mass spectrometry. However, this interaction lowers the melting temperature of APE1, which is consistent with a loss of structure in APE1, as measured by both differential scanning fluorimetry and circular dichroism. These results are consistent with other findings that E3330 concentrations of >100 μM are required to inhibit APE1’s endonuclease activity. To determine the role of E3330’s negatively charged carboxylate in redox inhibition, we converted the carboxylate to an amide by synthesizing (E)-2-[(4,5-dimethoxy-2-methyl-3,6-dioxocyclohexa-1,4-dien-1-yl)methylene]-N-methoxy-undecanamide (E3330-amide), a novel uncharged derivative. E3330-amide has no effect on the melting temperature of APE1, suggesting that it does not interact with the fully folded protein. However, E3330-amide inhibits APE1’s redox activity in in vitro electrophoretic mobility shift redox and cell-based transactivation assays, producing IC50 values (8.5 and 7 μM) lower than those produced with E3330 (20 and 55 μM, respectively). Thus, E3330’s negatively charged carboxylate is not required for redox inhibition. Collectively, our results provide additional support for a mechanism of redox inhibition involving interaction of E3330 or E3330-amide with partially unfolded APE1

Destabilisation, aggregation, toxicity and cytosolic mislocalisation of nucleophosmin regions associated with acute myeloid leukemia

Nucleophosmin (NPM1) is a multifunctional protein that is implicated in the pathogenesis of several human malignancies. To gain insight into the role of isolated fragments of NPM1 in its biological activities, we dissected the C-terminal domain (CTD) into its helical fragments. Here we focus the attention on the third helix of the NPM1-CTD in its wild-type (H3 wt) and AML-mutated (H3 mutA and H3 mutE) sequences. Conformational studies, by means of CD and NMR spectroscopies, showed that the H3 wt peptide was partially endowed with an a-helical structure, but the AML-sequences exhibited a lower content of this conformation, particularly the H3 mutA peptide. Thioflavin T assays showed that the H3 mutE and the H3 mutA peptides displayed a significant aggregation propensity that was confirmed by CD and DLS assays. In addition, we found that the H3 mutE and H3 mutA peptides, unlike the H3 wt, were moderately and highly toxic, respectively, when exposed to human neuroblastoma cells. Cellular localization experiments confirmed that the mutated sequences hamper their nucleolar accumulation, and more importantly, that the helical conformation of the H3 region is crucial for such a localization

Destabilisation, aggregation, toxicity and cytosolic mislocalisation of nucleophosmin regions associated with acute myeloid leukemia

Nucleophosmin (NPM1) is a multifunctional protein that is implicated in the pathogenesis of several human malignancies. To gain insight into the role of isolated fragments of NPM1 in its biological activities, we dissected the C-terminal domain (CTD) into its helical fragments. Here we focus the attention on the third helix of the NPM1-CTD in its wild-type (H3 wt) and AML-mutated (H3 mutA and H3 mutE) sequences. Conformational studies, by means of CD and NMR spectroscopies, showed that the H3 wt peptide was partially endowed with an a-helical structure, but the AML-sequences exhibited a lower content of this conformation, particularly the H3 mutA peptide. Thioflavin T assays showed that the H3 mutE and the H3 mutA peptides displayed a significant aggregation propensity that was confirmed by CD and DLS assays. In addition, we found that the H3 mutE and H3 mutA peptides, unlike the H3 wt, were moderately and highly toxic, respectively, when exposed to human neuroblastoma cells. Cellular localization experiments confirmed that the mutated sequences hamper their nucleolar accumulation, and more importantly, that the helical conformation of the H3 region is crucial for such a localization