Copper(II) complexes of the N‐terminal peptide fragments of tau protein have been studied by potentiometric and various spectroscopic techniques (UV‐vis, CD, ESR and ESI‐MS). The octapeptide Tau(9‐16) (Ac−EVMEDHAG−NH2) contains the H14 residue of the native protein, while Tau(26‐33) (Ac−QGGYTMHQ−NH2) and its mutants Tau(Q26K‐Q33K) (Ac−KGGYTMHK−NH2) and Tau(Q26K‐Y29A‐Q33K) (Ac−KGGATMHK−NH2) include the H32 residue. To compare the binding ability of H14 and H32 in a single molecule the decapeptide Ac−EDHAGTMHQD−NH2 (Tau(12‐16)(30‐34)) has also been synthesized and studied. The histidyl residue is the primary metal binding site for metal ions in all the peptide models studied. In the case of Tau(9‐16) the side chain carboxylate functions enhance the stability of the M−Nim coordinated complexes compared to Tau(26‐33) (logK(Cu−Nim)=5.04 and 3.78, respectively). Deprotonation and metal ion coordination of amide groups occur around the physiological pH range for copper(II). The formation of the imidazole‐ and amide‐coordinated species changes the metal ion preference and the complexes formed with the peptides containing the H32 residue predominate over those of H14 at physiological pH values (90 %–10 %) and in alkaline samples (96 %–4 %).
We report an ATP-dependent ubiquitin conjugation with IDE which, in turn, promotes Ub-Ub linkages in tube tests. We propose a novel function for IDE as a non-canonical ubiquitin activating enzyme.
Proliferation and programmed cell death are tightly correlated with the ubiquitin-proteasome system (UPS). Alterations in the UPS may be implicated in pathological conditions such as the proteasome over-activity in cancer cells. Mounting evidence indicates that many types of actively proliferating malignant cells are more sensitive to proteasome inhibition than normal cells, and therefore UPS inhibitors are actively pursued as anticancer agents. The approval of the proteasome inhibitor drug bortezomib for the treatment of myeloma and lymphoma further highlights the need for UPS inhibitors. Recent studies have suggested that clioquinol and 5-amino-8-hydroxyquinoline can inhibit proteasome activity and induce apoptosis in human cancer cells. As for clioquinol, a copper-dependent and -independent mechanism has been proposed to explain the inhibition of the proteasome whereas the activity of 5-amino-8-hydroxyquinoline has not been explored in the presence of copper(ii) ions. Herein, we investigated the biological activity of some 8-hydroxyquinolines by using human ovarian (A2780) and lung (A549) cancer cells. The effect of copper(ii) on the activity of these compounds was also evaluated. The investigated systems inhibit the chymotrypsin-like activity of the proteasome and induce growth inhibition and apoptosis in a concentration-dependent manner. Copper(ii) ions increase the activity of 8-hydroxyquinoline derivatives except in the case of 5-amino-8-hydroxyquinoline. This study suggests the great potential of amino- and chloro-8-hydroxyquinolines as anticancer agents. Furthermore, it clarifies some aspects concerning the activity of 5-amino-8-hydroxyquinoline, which has been previously proposed as a proteasome inhibitor capable of overcoming resistance to bortezomib.
Due to their altered metabolism cancer cells are more sensitive to proteasome inhibition or changes of copper levels than normal cells. Thus, the development of copper complexes endowed with proteasome inhibition features has emerged as a promising anticancer strategy. However, limited information is available about the exact mechanism by which copper inhibits proteasome. Here we show that Cu(II) ions simultaneously inhibit the three peptidase activities of isolated 20S proteasomes with potencies (IC50) in the micromolar range. Cu(II) ions, in cell-free conditions, neither catalyze red-ox reactions nor disrupt the assembly of the 20S proteasome but, rather, promote conformational changes associated to impaired channel gating. Notably, HeLa cells grown in a Cu(II)-supplemented medium exhibit decreased proteasome activity. This effect, however, was attenuated in the presence of an antioxidant. Our results suggest that if, on one hand, Cu(II)-inhibited 20S activities may be associated to conformational changes that favor the closed state of the core particle, on the other hand the complex effect induced by Cu(II) ions in cancer cells is the result of several concurring events including ROS-mediated proteasome flooding, and disassembly of the 26S proteasome into its 20S and 19S components.
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