RESUMO
In this study, an ethanol extract of Agave lechuguilla was evaluated against six carcinogenic cell lines (HCT-15, MCF-7, PC-3, U-251, SK-LU-1 and K-562) with an inhibition of 75.7 ± 2.3% against the SK-LU-1 line. Based on the previous result, the extract was hydrolyzed and fractionated, to which the IC50 was determined; the cell line was more sensitive to the fractionated extract with an IC50 6.96 ± 0.15 µg/mL. Characterization by mass spectrometry showed the presence of kaempferol, quercetin and a flavonoid dimer formed by afzelechin-4ß-8-quercetin, according to the generated fragmentation pattern. The fractionated extract presented cell death by apoptosis with 39.8% at 24 h. Molecular docking was performed with the molecules found to try to describe cell death by apoptosis through death receptors such as FasCD95, TNF-R1, DR4/5 and blocking signaling on the EGFR and K-Ras MAPK/ERK pathway, as well as through the intrinsic pathway activating tBID, which promotes the amplification of the apoptotic signal due to the activation of caspase-3, and consequently caspase-7. In addition to the activation of the IIb complex associated with cell death due to necroptosis.
Assuntos
Agave/química , Antineoplásicos Fitogênicos/química , Antineoplásicos Fitogênicos/farmacologia , Apoptose/efeitos dos fármacos , Extratos Vegetais/química , Extratos Vegetais/farmacologia , Adenocarcinoma de Pulmão , Antioxidantes/química , Antioxidantes/farmacologia , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Humanos , Ligação de Hidrogênio , Modelos Moleculares , Conformação Molecular , Estrutura Molecular , Compostos Fitoquímicos/química , Compostos Fitoquímicos/farmacologia , Relação Estrutura-AtividadeRESUMO
The CFIm25 subunit of the heterotetrameric cleavage factor Im (CFIm) is a critical factor in the formation of the poly(A) tail at mRNA 3' end, regulating the recruitment of polyadenylation factors, poly(A) site selection, and cleavage/polyadenylation reactions. We previously reported the homologous protein (EhCFIm25) in Entamoeba histolytica, the protozoan causing human amoebiasis, and showed the relevance of conserved Leu135 and Tyr236 residues for RNA binding. We also identified the GUUG sequence as the recognition site of EhCFIm25. To understand the interactions network that allows the EhCFIm25 to maintain its three-dimensional structure and function, here we performed molecular dynamics simulations of wild-type (WT) and mutant proteins, alone or interacting with the GUUG molecule. Our results indicated that in the presence of the GUUG sequence, WT converged more quickly to lower RMSD values in comparison with mutant proteins. However, RMSF values showed that movements of amino acids of WT and EhCFIm25*L135 T were almost identical, interacting or not with the GUUG molecule. Interestingly, EhCFIm25*L135 T, which is the only mutant with a slight RNA binding activity experimentally, presents the same stabilization of bend structures and alpha helices as WT, notably in the C-terminus. Moreover, WT and EhCFIm25*L135 T presented almost the same number of contacts that mainly involve lysine residues interacting with the G4 nucleotide. Overall, our data proposed a clear description of the structural and mechanistic data that govern the RNA binding capacity of EhCFIm25.
Assuntos
Entamoeba histolytica/química , Leucina/química , Proteínas de Protozoários/química , RNA Bacteriano/química , RNA Mensageiro/química , Tirosina/química , Fatores de Poliadenilação e Clivagem de mRNA/química , Substituição de Aminoácidos , Sítios de Ligação , Cristalografia por Raios X , Entamoeba histolytica/genética , Entamoeba histolytica/metabolismo , Leucina/metabolismo , Simulação de Dinâmica Molecular , Mutação , Poli A/química , Poli A/genética , Poli A/metabolismo , Ligação Proteica , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Domínios e Motivos de Interação entre Proteínas , Multimerização Proteica , Proteínas de Protozoários/genética , Proteínas de Protozoários/metabolismo , RNA Bacteriano/genética , RNA Bacteriano/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Termodinâmica , Tirosina/metabolismo , Fatores de Poliadenilação e Clivagem de mRNA/genética , Fatores de Poliadenilação e Clivagem de mRNA/metabolismoRESUMO
Bax is a member of the Bcl-2 protein family that participates in mitochondrion-mediated apoptosis. In the early stages of the apoptotic pathway, this protein migrates from the cytosol to the outer mitochondrial membrane, where it is inserted and usually oligomerizes, making cytochrome c-compatible pores. Although several cellular and structural studies have been reported, a description of the stability of Bax at the molecular level remains elusive. This article reports molecular dynamics simulations of monomeric Bax at 300, 400, and 500 K, focusing on the most relevant structural changes and relating them to biological experimental results. Bax gradually loses its α-helices when it is submitted to high temperatures, yet it maintains its globular conformation. The resistance of Bax to adopt an extended conformation could be due to several interactions that were found to be responsible for maintaining the structural stability of this protein. Among these interactions, we found salt bridges, hydrophobic interactions, and hydrogen bonds. Remarkably, salt bridges were the most relevant to prevent the elongation of the structure. In addition, the analysis of our results suggests which conformational movements are implicated in the activation/oligomerization of Bax. This atomistic description might have important implications for understanding the functionality and stability of Bax in vitro as well as within the cellular environment.