RESUMEN
Mitochondrial targeting is a promising approach for solving current issues in clinical application of chemotherapy and diagnosis of several disorders. Here, we discuss direct conjugation of mitochondrial-targeting moieties to anticancer drugs, antioxidants and sensor molecules. Among them, the most widely applied mitochondrial targeting moiety is triphenylphosphonium (TPP), which is a delocalized cationic lipid that readily accumulates and penetrates through the mitochondrial membrane due to the highly negative mitochondrial membrane potential. Other moieties, including short peptides, dequalinium, guanidine, rhodamine, and F16, are also known to be promising mitochondrial targeting agents. Direct conjugation of mitochondrial targeting moieties to anticancer drugs, antioxidants and sensors results in increased cytotoxicity, anti-oxidizing activity and sensing activity, respectively, compared with their non-targeting counterparts, especially in drug-resistant cells. Although many mitochondria-targeted anticancer drug conjugates have been investigated in vitro and in vivo, further clinical studies are still needed. On the other hand, several mitochondria-targeting antioxidants have been analyzed in clinical phases I, II and III trials, and one conjugate has been approved for treating eye disease in Russia. There are numerous ongoing studies of mitochondria-targeted sensors.
RESUMEN
The main aim of this study was to obtain porous antimicrobial composites consisting of chitosan, alginate, and biosynthesized silver nanoparticles (AgNPs). Chitosan and alginate were used owing to their pore-forming capacity, while AgNPs were used for their antimicrobial property. The developed porous composites of chitosan-alginate-AgNPs were characterized using Fourier transform infrared spectroscopy (FT-IR), ultraviolet-visible spectroscopy, X-ray diffraction (XRD) analysis, and scanning electron microscopy (SEM). The FT-IR results revealed the presence of a strong chemical interaction between chitosan and alginate due to polyelectrolyte complex; whereas, the XRD results confirmed the presence of AgNPs in the composites. The dispersion of AgNPs in the porous membrane was uniform with a pore size of 50-500µm. Antimicrobial activity of the composites was checked with Escherichia coli and Staphylococcus aureus. The developed composites resulted in the formation of a zone of inhibition of 11±1mm for the Escherichia coli, and 10±1mm for Staphylococcus aureus. The bacterial filtration efficiency of chitosan-alginate-AgNPs was 1.5-times higher than that of the chitosan-alginate composite. The breast cancer cell line MDA-MB-231 was used to test the anticancer activity of the composites. The IC50 value of chitosan-alginate-AgNPs on MDA-MB-231 was 4.6mg. The developed chitosan-alginate-AgNPs composite showed a huge potential for its applications in antimicrobial filtration and cancer treatment.
Asunto(s)
Alginatos/química , Quitosano/química , Nanopartículas del Metal/química , Plata/química , Plata/farmacología , Antibacterianos/síntesis química , Antibacterianos/química , Antibacterianos/farmacología , Antineoplásicos/síntesis química , Antineoplásicos/química , Antineoplásicos/farmacología , Apoptosis/efectos de los fármacos , Línea Celular Tumoral , Escherichia coli/efectos de los fármacos , Ácido Glucurónico/química , Ácidos Hexurónicos/química , Humanos , Pruebas de Sensibilidad Microbiana , Porosidad , Staphylococcus aureus/efectos de los fármacosRESUMEN
Peptidyl prolyl cis/trans isomerases (PPIases) catalyze the cis/trans isomerization of peptidyl-prolyl peptide bonds preceding prolines. We investigated the protein-protein interaction between a 22 kDa PPIase (VaFKBP22, an FK506-binding protein) and the molecular chaperone DnaK derived from Vibrio anguillarum O1 (VaDnaK) using GST pull-down assays and a bacterial two-hybrid system for in vivo and in vitro studies, respectively. Furthermore, we analyzed the three-dimensional structure of the protein-protein interaction. Based on our results, VaFKBP22 appears to act as a cochaperone of VaDnaK, and contributes to protein folding and stabilization via its peptidyl-prolyl cis/trans isomerization activity.
Asunto(s)
Proteínas Bacterianas/metabolismo , Proteínas de Choque Térmico/metabolismo , Isomerasa de Peptidilprolil/metabolismo , Vibrio/metabolismo , Proteínas Bacterianas/química , Sitios de Unión , Proteínas de Choque Térmico/química , Modelos Moleculares , Peso Molecular , Isomerasa de Peptidilprolil/química , Unión Proteica , Conformación Proteica , Dominios y Motivos de Interacción de Proteínas , Mapeo de Interacción de Proteínas , Multimerización de ProteínaRESUMEN
Peptidyl-prolyl cis/trans isomerase (PPIase) catalyzes the isomerization of peptide bonds to achieve conformational changes in native folded proteins. An FKBP-type PPIase with an approximate molecular weight of 17kDa was isolated from Vibrio anguillarum O1 and named VaFKBP17. To investigate its biochemical properties, the ppi gene from V. anguillarum O1 was isolated and overexpressed in Escherichia coli. A protease-coupled assay for isomerization activity, using Succinyl-Ala-Phe-Pro-Phe-p nitroanilide as substrate, indicated that the activity of VaFKBP17 was highest at low temperature (5°C) and alkaline conditions (pH 10). The immunosuppressant FK506 inhibited the isomerization activity of VaFKBP17. The chaperone activity of VaFKBP17 was assessed using a citrate synthase thermal aggregation activity assay. To evaluate its ability to catalyze protein refolding, the effect of VaFKBP17 on inclusion bodies was investigated during a dilution process. In this assay, VaFKBP17 was able to assist protein refolding. These results provide evidence that VaFKBP17 possesses chaperone-like activity. The structural homology of VaFKBP17 relative to other known bacterial FKBPs was also examined.
Asunto(s)
Proteínas Bacterianas/aislamiento & purificación , Isomerasa de Peptidilprolil/aislamiento & purificación , Plásmidos/metabolismo , Proteínas Recombinantes de Fusión/aislamiento & purificación , Vibrio/química , Secuencia de Aminoácidos , Proteínas Bacterianas/antagonistas & inhibidores , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Clonación Molecular , Escherichia coli/genética , Escherichia coli/metabolismo , Expresión Génica , Histidina/química , Histidina/genética , Concentración de Iones de Hidrógeno , Cuerpos de Inclusión/química , Cuerpos de Inclusión/enzimología , Modelos Moleculares , Datos de Secuencia Molecular , Peso Molecular , Oligopéptidos/química , Oligopéptidos/genética , Isomerasa de Peptidilprolil/antagonistas & inhibidores , Isomerasa de Peptidilprolil/química , Isomerasa de Peptidilprolil/genética , Plásmidos/química , Pliegue de Proteína , Estructura Terciaria de Proteína , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/genética , Alineación de Secuencia , Homología Estructural de Proteína , Especificidad por Sustrato , Tacrolimus/química , Temperatura , Vibrio/enzimologíaRESUMEN
Vibrio anguillarum, a causative agent of vibriosis in finfish, crustaceans, and bivalves, is a Gram-negative, motile marine bacterium. Most bacteria have developed survival strategies in various environments. The aim of this study was to investigate the changes in protein expression of V. anguillarum O1 incubated under different conditions using two dimensional electrophoresis and MALDI-TOF MS/MS analysis. Result indicated that peptidyl-prolyl cis/trans isomerase (PPIase) expression was increasingly appeared when incubated at low temperature (15°C) and alkaline conditions (pH 10). Subsequently, the ppi gene from V. anguillarum O1 was isolated and overexpressed in Escherichia coli to characterize the biochemical properties. The cloned ppi gene encoded 206 amino acids containing the conserved regions identified in FK506 binding pocket. To determine the optimal conditions of the purified recombinant PPIase protein (VaFKBP22), we used Succinyl-Ala-Phe-Pro-Phe-p nitroanilide as substrate and the highest enzymatic activity was found at 5°C and pH 6. VaFKBP22 was detected in the cytoplasm and periplasm of V. anguillarum O1. In addition, VaFKBP22 also showed chaperone activity and did not show cytotoxic activity.