RESUMO
Amyloid ß (Aß) oligomers are the most neurotoxic forms of Aß, and Aß(1-42) is the prevalent Aß peptide found in the amyloid plaques of Alzheimer's disease patients. Aß(25-35) is the shortest peptide that retains the toxicity of Aß(1-42). Aß oligomers bind to calmodulin (CaM) and calbindin-D28k with dissociation constants in the nanomolar Aß(1-42) concentration range. Aß and histidine-rich proteins have a high affinity for transition metal ions Cu2+, Fe3+ and Zn2+. In this work, we show that the fluorescence of Aß(1-42) HiLyteTM-Fluor555 can be used to monitor hexa-histidine peptide (His6) interaction with Aß(1-42). The formation of His6/Aß(1-42) complexes is also supported by docking results yielded by the MDockPeP Server. Also, we found that micromolar concentrations of His6 block the increase in the fluorescence of Aß(1-42) HiLyteTM-Fluor555 produced by its interaction with the proteins CaM and calbindin-D28k. In addition, we found that the His6-tag provides a high-affinity site for the binding of Aß(1-42) and Aß(25-35) peptides to the human recombinant cytochrome b5 reductase, and sensitizes this enzyme to inhibition by these peptides. In conclusion, our results suggest that a His6-tag could provide a valuable new tool to experimentally direct the action of neurotoxic Aß peptides toward selected cellular targets.
Assuntos
Doença de Alzheimer , Peptídeos beta-Amiloides , Humanos , Peptídeos beta-Amiloides/metabolismo , Histidina/química , Hexosaminidase A , Calbindina 1 , Cobre/química , Fragmentos de Peptídeos/química , Doença de Alzheimer/metabolismoRESUMO
Hydrogen sulfide (H(2)S) is an endogenously generated gas that can also be administered exogenously. It modulates physiological functions and has reported cytoprotective effects. To evaluate a possible antioxidant role, we investigated the reactivity of hydrogen sulfide with several one- and two-electron oxidants. The rate constant of the direct reaction with peroxynitrite was (4.8±1.4)×10(3)M(-1) s(-1) (pH 7.4, 37°C). At low hydrogen sulfide concentrations, oxidation by peroxynitrite led to oxygen consumption, consistent with a one-electron oxidation that initiated a radical chain reaction. Accordingly, pulse radiolysis studies indicated that hydrogen sulfide reacted with nitrogen dioxide at (3.0±0.3)×10(6)M(-1) s(-1) at pH 6 and (1.2±0.1)×10(7)M(-1) s(-1) at pH 7.5 (25°C). The reactions of hydrogen sulfide with hydrogen peroxide, hypochlorite, and taurine chloramine had rate constants of 0.73±0.03, (8±3)×10(7), and 303±27M(-1) s(-1), respectively (pH 7.4, 37°C). The reactivity of hydrogen sulfide was compared to that of low-molecular-weight thiols such as cysteine and glutathione. Considering the low tissue concentrations of endogenous hydrogen sulfide, direct reactions with oxidants probably cannot completely account for its protective effects.
Assuntos
Sulfeto de Hidrogênio/metabolismo , Oxidantes/metabolismo , Ácido Peroxinitroso/metabolismo , Catálise , Avaliação de Medicamentos , Peróxido de Hidrogênio/metabolismo , Peróxido de Hidrogênio/farmacologia , Sulfeto de Hidrogênio/farmacologia , Ácido Hipocloroso/farmacologia , Técnicas In Vitro , Dióxido de Nitrogênio/metabolismo , Oxirredução , Estresse Oxidativo/fisiologia , Oxigênio/metabolismo , Taurina/análogos & derivados , Taurina/metabolismo , Taurina/farmacologiaRESUMO
Selective enzymatic hydrolysis, lipid compositional analyses, and fluorescence studies have been carried out on acetylcholine receptor (AChR)-rich membranes from Torpedinidae to investigate the topology of sphingomyelin (SM) in the native membrane and its relationship with the AChR protein. Controlled sphingomyelinase hydrolysis of native membranes showed that SM is predominantly (approximately 60%) localized in the outer half of the lipid bilayer. Differences were also observed in the distribution of SM fatty acid molecular species in the two bilayer leaflets. A fluorescent SM derivative ( N-[10-(1-pyrenyl)decanoyl]sphingomyelin; Py-SM) was used to study protein-lipid interactions in the AChR-rich membrane and in affinity-purified Torpedo AChR reconstituted in liposomes made from Torpedo electrocyte lipid extracts. The efficiency of Förster resonance energy transfer (FRET) from the protein to the pyrenyl-labeled lipid as a function of acceptor surface density was used to estimate distances and topography of the SM derivative relative to the protein. The dynamics of the lipid acyl chains were explored by measuring the thermal dependence of Py-SM excimer formation, sensitive to the fluidity of the membrane. Differences were observed in the concentration dependence of excimer/monomer pyrenyl fluorescence when measured by direct excitation of the probe as against under FRET conditions, indicating differences in the intermolecular collisional frequency of the fluorophores between bulk and protein-vicinal lipid environments, respectively. Py-SM exhibited a moderate selectivity for the protein-vicinal lipid domain, with a calculated relative affinity K(r) approximately 0.55. Upon sphingomyelinase digestion of the membrane, FRET efficiency increased by about 50%, indicating that the resulting pyrenyl-ceramide species have higher affinity for the protein than the parental SM derivative.