Stability constants and ligand donor sets of the copper(II) complexes of the NH₂-29–56(L¹)(AA³⁰GKTKEGVLYV⁴⁰GSKTKEGVVH⁵⁰GVATVA⁵⁶-NH₂), NH₂-M²⁹-D³⁰-56(L²) and Ac-M²⁹-D³⁰-56(L³) fragments of α-synuclein were determined in aqueous solution for 1 : 1 metal-to-ligand molar ratio in the pH range 2.5–10.5. The tyrosine residue in the 39th position of the α-synuclein fragments does not take part in the coordination of the metal ion. The potentiometric and spectroscopic data (UV-Vis, CD, EPR) show that acetylation of the amino terminal group induces significant changes in the coordination properties of the L³ fragment compared to that of the L² peptide. When the amino group is blocked (L³) the imidazole nitrogen of the histidine residue acts as an anchoring site and at higher pH the 3N {N_Im,2N⁻} and 4N {N_Im,3N⁻} complexes are formed. The L¹ peptide at physiological pH forms in equilibrium 3N {NH₂,N⁻,CO,N_Im} and 4N {NH₂,2N⁻,N_Im} complexes. For the L² peptide the coordination of the copper(II) ions starts from the N-terminal Met residue and with increasing of pH the Asp residue in second position of amino acid sequence coordinates and stabilizes significantly the 2N complex as a result of chelation through the β-carboxylate group. At physiological pH the 3N {NH₂,N⁻,β-COO⁻,N_Im} coordination mode dominates. At pH above 6 the results for the L² fragment suggest the formation of 3N and 4N complexes (in equatorial plane) and the involvement of the lateral NH₂ group of Lys residue in the axial coordination of Cu(II) ion. In CD spectra σ (ε-NH₂-Lys) → Cu(II) charge transfer transition is observed. The stability constants for the L² fragment of α-synuclein of the 4N {NH₂,2N⁻,N_Im} and {NH₂,3N⁻} complexes are higher about 1.5 and 0.7 orders of magnitude, respectively, by comparison to those of the L¹ peptide. This increase may be explained by the involvement of the ε-NH₂ group of Lys residue in the coordination sphere of metal ion.

Proline- and arginine-rich peptide PR11 is an allosteric inhibitor of 20S proteasome. We modified its sequence inter alia by introducing HbYX, RYX, or RHbX C-terminal extensions (Hb, hydrophobic moiety; R, arginine; Y, tyrosine; X, any residue). Consequently, we were able to improve inhibitory potency or to convert inhibitors into strong activators: the former with an aromatic penultimate Hb residue and the latter with the HbYX motif. The PR peptide activator stimulated 20S proteasome in vitro to efficiently degrade protein substrates, such as α-synuclein and enolase, but also activated proteasome in cultured fibroblasts. The positive and negative PR modulators differently influenced the proteasome conformational dynamics and affected opening of the substrate entry pore. The resolved crystal structure showed PR inhibitor bound far from the active sites, at the proteasome outer face, in the pocket used by natural activators. Our studies indicate the opportunity to tune proteasome activity by allosteric regulators based on PR peptide scaffold.

Earthworm coelomic fluid (CF) is known as a rich source of various bioactive compounds with promising anticancer features. However, it has been demonstrated that CF affects functionality of both, cancer and normal cells. This non-selective activity causes a major problem for medical application of CF. In this study, we present the anticancer activity of the active protein-carbohydrate fraction (AF) isolated from thermally treated CF of earthworm Dendrobaena veneta. The in vitro effect of the AF was examined in human colon model including normal human colon epithelium (CCD 841 CoTr) and human colon adenocarcinoma (HT-29 and LS180) cell lines. We investigated the impact of AF on cell viability neutral red and lactate dehydrogenase assays, morphology May-Grünwald-Giemsa staining assay proliferation MTT tetrazolium salt and BrdU incorporation assays as well as cell cycle progression propidium iodide/RNase staining and the activity of human 20S proteasome the hydrolysis of AMC from a Suc-LLVY-AMC peptide substrate. Additionally, the influence of AF on apoptosis was examined in HT-29 cells by Annexin V/PI, Hoechst 33342 staining and active caspase-3 assays. Our investigation demonstrated that AF at the tested concentration range does not affect the viability and morphology of CCD 841 CoTr cells. Simultaneously, AF inhibits human 20S proteasome activity as well as significantly decreases mitochondrial metabolism, disturbs cell cycle and induces apoptosis via activation of procaspase-3 in HT-29 cancer cells. Obtained results demonstrate the antiproliferative and proapoptotic activity of AF that can be useful in developing therapeutic strategies to treat human colon cancer.