RESUMO
A cholesteryl-functionalized derivative of activity dependent neurotrophic factor-9 peptide (a nine amino acid core peptide of activity-dependent neurotrophic factor, acting against Alzheimer's disease) was synthesized aiming at the improvement of its bioavailability. Therefore, its uptake was comparatively investigated with that of its parent peptide by employing mouse neuroblastoma Neuro-2a cells. Owing to the hydrophobic character of this cholesteryl-functionalized peptide, it exhibited enhanced permeability and intracellular uptake while it also retained its low cytotoxicity at concentrations up to 1 µM. FACS analysis also revealed that when Neuro-2a cells were treated with this activity dependent neurotrophic factor-9 derivative, at a concentration of 50 nM, an almost 100% uptake was obtained. In addition, in vitro biological activity experiments showed that the functionalized peptide retained its neurotrophic activity at femtomolar concentration range.
Assuntos
Neuropeptídeos/química , Fármacos Neuroprotetores/química , Animais , Linhagem Celular Tumoral , Permeabilidade da Membrana Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Fluoresceína-5-Isotiocianato/química , Interações Hidrofóbicas e Hidrofílicas , Camundongos , N-Metilaspartato/química , N-Metilaspartato/toxicidade , Neuropeptídeos/síntese química , Neuropeptídeos/farmacologia , Fármacos Neuroprotetores/síntese química , Fármacos Neuroprotetores/farmacologia , OligopeptídeosRESUMO
Sco1 and Cox17 are accessory proteins required for the correct assembly of eukaryotic cytochrome c oxidase. At variance with Sco1, Cox17 orthologs are found only in eukaryotes. We browsed bacterial genomes to search proteins functionally equivalent to Cox17, and we identified a class of proteins of unknown function displaying a conserved gene neighborhood to bacterial Sco1 genes, all sharing a potential metal binding motif H(M)X10MX21HXM. Two members of this group, DR1885 from Deinococcus radiodurans and CC3502 from Caulobacter crescentus, were expressed, and their interaction with copper was investigated. The solution structure and extended x-ray absorption fine structure data on the former protein reveal that the protein binds copper(I) through a histidine and three Mets in a cupredoxin-like fold. The surface location of the copper-binding site as well as the type of coordination are well poised for metal transfer chemistry, suggesting that DR1885 might transfer copper, taking the role of Cox17 in bacteria. On the basis of our results, a possible pathway for copper delivery to the Cu(A) center in bacteria is proposed.
Assuntos
Proteínas de Bactérias/metabolismo , Caulobacter/metabolismo , Cobre/metabolismo , Deinococcus/metabolismo , Complexo IV da Cadeia de Transporte de Elétrons/biossíntese , Sequência de Aminoácidos , Proteínas de Bactérias/genética , Proteínas de Transporte , Proteínas de Transporte de Cátions/genética , Proteínas de Transporte de Cátions/metabolismo , Caulobacter/genética , Proteínas de Transporte de Cobre , Deinococcus/genética , Dados de Sequência Molecular , Estrutura Terciária de ProteínaRESUMO
The human metallochaperone HAH1 has been produced in Escherichia coli with four additional amino acids at the C-terminus and characterized in solution by NMR spectroscopy, both with and without copper(I). The solution structure of the apo-HAH1 monomer has a root-mean-square-deviation (RMSD) of 0.50 A for the coordinates of the backbone atoms and 0.96 A for all heavy atoms. These values compare, respectively, with 0.45 and 0.95 A for copper(I)-HAH1. There are only minor structural rearrangements upon copper(I) binding. In particular, the variation of interatomic interactions around the metal-binding region is limited to a movement of Lys60 toward the metal site. The protein structures are similar to those obtained by X-ray crystallography in a variety of derivatives, with backbone RMSD values below 1 A. In the holoprotein, copper(I) is confirmed to be two coordinated. If these data are compared with those of orthologue proteins, we learn that HAH1 has a lower tendency to change coordination number from two to three. Such a switch in coordination is a key step in copper transfer.
