ABSTRACT
Fasciclins (FAS1) are ancient adhesion protein domains with no common small ligand binding reported. A unique microalgal FAS1-containing astaxanthin (AXT)-binding protein (AstaP) binds a broad repertoire of carotenoids by a largely unknown mechanism. Here, we explain the ligand promiscuity of AstaP-orange1 (AstaPo1) by determining its NMR structure in complex with AXT and validating this structure by SAXS, calorimetry, optical spectroscopy and mutagenesis. α1-α2 helices of the AstaPo1 FAS1 domain embrace the carotenoid polyene like a jaw, forming a hydrophobic tunnel, too short to cap the AXT ß-ionone rings and dictate specificity. AXT-contacting AstaPo1 residues exhibit different conservation in AstaPs with the tentative carotenoid-binding function and in FAS1 proteins generally, which supports the idea of AstaP neofunctionalization within green algae. Intriguingly, a cyanobacterial homolog with a similar domain structure cannot bind carotenoids under identical conditions. These structure-activity relationships provide the first step towards the sequence-based prediction of the carotenoid-binding FAS1 members.
Subject(s)
Carrier Proteins , Cell Adhesion Molecules , Ligands , Scattering, Small Angle , X-Ray Diffraction , Cell Adhesion Molecules/metabolism , Carotenoids/metabolismABSTRACT
In recent years, new prospects for the use of nucleic acids as anticancer drugs have been discovered. Aptamers for intracellular targets can regulate cellular functions and cause cell death or proliferation. However, intracellular aptamers have limited use for therapeutic applications due to their low bioavailability. In this work, we selected DNA aptamers to cell organelles and nucleus of cancer cells, and showed that an aptamer NAS-24 binds to vimentin and causes apoptosis of mouse ascites adenocarcinoma cells in vitro and in vivo. To deliver the aptamer NAS-24 inside cells, natural polysaccharide arabinogalactan was used as a carrier reagent. The mixture of arabinogalactan and NAS-24 was injected intraperitonealy for 5 days into mice with adenocarcinoma and inhibited adenocarcinoma growth more effectively than free arabinogalactan or the aptamer alone. The use of aptamers to intracellular targets together with arabinogalactan becomes a promising approach for anticancer therapy.
Subject(s)
Adenocarcinoma/therapy , Aptamers, Nucleotide/genetics , Carcinoma, Ehrlich Tumor/therapy , Gene Expression Regulation, Neoplastic , Neoplasm Proteins/genetics , Vimentin/genetics , Adenocarcinoma/genetics , Adenocarcinoma/metabolism , Adenocarcinoma/pathology , Animals , Antineoplastic Agents/chemical synthesis , Antineoplastic Agents/metabolism , Apoptosis , Aptamers, Nucleotide/metabolism , Carcinoma, Ehrlich Tumor/genetics , Carcinoma, Ehrlich Tumor/metabolism , Carcinoma, Ehrlich Tumor/pathology , Cell Line, Tumor , Drug Carriers/chemistry , Drug Carriers/isolation & purification , Galactans/chemistry , Galactans/isolation & purification , Genetic Therapy , Hepatocytes/metabolism , Hepatocytes/pathology , Humans , Larix/chemistry , Liver/metabolism , Liver/pathology , Male , Mice , Mice, Inbred ICR , Molecular Targeted Therapy/methods , Neoplasm Proteins/antagonists & inhibitors , Neoplasm Proteins/metabolism , Vimentin/antagonists & inhibitors , Vimentin/metabolismABSTRACT
Salmonella is one of the most dangerous and common food-borne pathogens. The overuse of antibiotics for disease prevention has led to the development of multidrug resistant Salmonella. Now, more than ever, there is a need for new antimicrobial drugs to combat these resistant bacteria. Aptamers have grown in popularity since their discovery, and their properties make them attractive candidates for therapeutic use. In this work, we describe the selection of highly specific DNA aptamers to S. enteritidis and S. typhimurium. To evolve species-specific aptamers, twelve rounds of selection to live S. enteritidis and S. typhimurium were performed, alternating with a negative selection against a mixture of related pathogens. Studies have shown that synthetic pools combined from individual aptamers have the capacity to inhibit growth of S. enteritidis and S. typhimurium in bacterial cultures; this was the result of a decrease in their membrane potential.
