RESUMO
The cholesterol-ester transfer protein (CETP) exchanges lipids between high-density lipoproteins (HDLs) and low-density lipoproteins (LDLs). The excessive transport of lipids from HDLs to LDLs mediated by this protein can cause an alteration in the deposition of lipoproteins onto the arterial walls, thus promoting the development of arteriosclerosis. Different CETP inhibitors have been tested in recent years, but none has been confirmed as being effectively palliative for the disease. We employed in silico databases and molecular docking as a computational method to predict how potential CETP inhibitors could interact with the active site of the CETP protein. Upon previously comparing two computer software packages to determine which generated a greater number of accurate CETP-inhibitor-complex structures, we chose the more appropriate program for our studies. We then abstracted a series of databases of known CETP inhibitors and noninhibitors exhibiting different 50% concentrations of CETP-inhibitory (INH) activity, to generate virtual structures for docking with different combinations of the CETP receptor. From this process, we obtained as the most suitable structure 4F2A_1OB_C_PCW-it accordingly having a greater area under the receiver operating characteristic curve. The molecular docking of known compounds in comparison with the respective conformation of this inhibitor enabled us to obtain ΔGs (in kcal/mol) from which data we made a first exploration of unknown compounds for CETP-INH activity. Thus, the 4F2A_1OB_C_PCW structure was docked with DrugBank-Approved commercial compounds in an extensive database, whose status had already been established from pharmacokinetics and toxicology. In this study, we present a group of potential compounds as CETP-inhibitor candidates.
Assuntos
Proteínas de Transferência de Ésteres de Colesterol/antagonistas & inibidores , Compostos de Anilina/farmacologia , Área Sob a Curva , Soluções Tampão , Cristalização , Bases de Dados de Compostos Químicos , Humanos , Concentração Inibidora 50 , Ligantes , Simulação de Acoplamento Molecular , Propanolaminas/farmacologia , Quinolinas/farmacologia , Curva ROC , SoroRESUMO
Discoidal high-density lipoproteins (D-HDL) are critical intermediates in reverse cholesterol transport. Most of the present knowledge of D-HDL is based on studies with reconstituted lipoprotein complexes of apolipoprotein A-I (apoA-I) obtained by cholate dialysis (CD). D-HDL can also be generated by the direct microsolubilization (DM) of phospholipid vesicles at the gel/fluid phase transition temperature, a process mechanistically similar to the "in vivo" apoAI lipidation via ABCA1. We compared the apoA-I configuration in D-HDL reconstituted with dimyristoylphosphatidylcholine by both procedures using fluorescence resonance energy transfer measurements with apoA-I tryptophan mutants and fluorescently labeled cysteine mutants. Results indicate that apoA-I configuration in D-HDL depends on the reconstitution process and are consistent with a "double belt" molecular arrangement with different helix registry. As reported by others, a configuration with juxtaposition of helices 5 of each apoAI monomer (5/5 registry) predominates in D-HDL obtained by CD. However, a configuration with helix 5 of one monomer juxtaposed with helix 2 of the other (5/2 registry) would predominate in D-HDL generated by DM. Moreover, we also show that the kinetics of cholesterol efflux from macrophage cultures depends on the reconstitution process, suggesting that apoAI configuration is important for this HDL function.
Assuntos
Apolipoproteína A-I/metabolismo , Colesterol/metabolismo , Lipoproteínas/metabolismo , Substituição de Aminoácidos , Animais , Apolipoproteína A-I/química , Apolipoproteína A-I/genética , Linhagem Celular , Colesterol/química , Colesterol/genética , Dimiristoilfosfatidilcolina/química , Dimiristoilfosfatidilcolina/metabolismo , Humanos , Lipoproteínas/química , Lipoproteínas/genética , Macrófagos/citologia , Macrófagos/metabolismo , Camundongos , Mutação de Sentido Incorreto , Estrutura Secundária de Proteína , Estrutura Terciária de ProteínaRESUMO
Apolipoprotein A-I (apoA-I) is the major protein component of high density lipoproteins. This protein has key functions in lipoprotein metabolism and its plasma concentration is inversely correlated with the incidence of atherosclerosis and cardiovascular diseases. There is an increasing need to develop methods for efficient production of recombinant apoA-I for using it in basic research or pharmacological therapy. An apoA-I variant lacking two amino acid residues at the N-terminus can be easily produced by bacterial expression. We report here the characterization of this variant comparing its properties with those of the protein isolated from human serum. The results validate the use of this variant in future assays and investigations.
Assuntos
Apolipoproteína A-I/química , Apolipoproteína A-I/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Apolipoproteína A-I/genética , Escherichia coli/genética , Escherichia coli/metabolismo , Guanidina , Humanos , Desnaturação Proteica , Estabilidade Proteica , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Reprodutibilidade dos Testes , TermodinâmicaRESUMO
Previous results [J. Biol. Chem. 276 (2001) 16978] indicated that an apolipoprotein A-I (apoAI) central region swings away from lipid contact in discoidal high density lipoproteins (HDL), but it is able to penetrate into the bilayer of lipid vesicles. In this work, we have studied the interaction with lipid membranes of a synthetic peptide with the sequence of apoAI region between residues 77 and 120 (AI 77-120). Like apoAI, AI 77-120 binds to phospholipid vesicles and shows selectivity for cholesterol-containing membranes. Moreover, AI 77-120 promotes cholesterol desorption from membranes in a similar fashion as apoAI and can stimulate cholesterol efflux from Chinese hamster ovary cells. AI 77-120 has a considerable alpha-helical content in water solution, and its secondary structure is not largely modified after binding to membranes. Both apoA-I and AI 77-120 are oligomeric in the lipid-bound state, suggesting that dimerization of the central domain could be required for the membrane binding activity of apoA-I in HDL.