RESUMEN
Many laboratory strains of Escherichia coli are resistant to methotrexate (MTX), a folate analogue that binds dihydrofolate reductase (DHFR). Mutations that inactivate either tolC or acrA confer MTX sensitivity. Further, overexpression of a fusion protein with DHFR activity reverses this sensitivity by titrating out intracellular MTX. These results suggest that MTX accumulates in cells where mutations in acrA or tolC have inactivated the TolC-dependent AcrAB multidrug resistance efflux pump.
Asunto(s)
Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/metabolismo , Proteínas Bacterianas/metabolismo , Inhibidores Enzimáticos/farmacología , Proteínas de Escherichia coli , Escherichia coli/efectos de los fármacos , Lipoproteínas/metabolismo , Metotrexato/farmacología , Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/genética , Proteínas de la Membrana Bacteriana Externa/metabolismo , Proteínas Bacterianas/genética , Transporte Biológico , Farmacorresistencia Microbiana/fisiología , Escherichia coli/enzimología , Escherichia coli/metabolismo , Antagonistas del Ácido Fólico/farmacología , Lipoproteínas/genética , Proteínas de Transporte de Membrana , Mutación , Tetrahidrofolato Deshidrogenasa/metabolismoRESUMEN
BACKGROUND: Chemically induced dimerization (CID) can be used to manipulate cellular regulatory pathways from signal transduction to transcription, and to create model systems for study of the specific interactions between proteins and small-molecule chemical ligands. However, few CID systems are currently available. The properties of, and interactions between, Escherichia coli dihydrofolate reductase (DHFR) and the ligand methotrexate (MTX) meet many of the desired criteria for the development of a new CID system. RESULTS: BisMTX, a homobifunctional version of MTX, was synthesized and tested for its ability to induce dimerization of DHFR. Gel-filtration analysis of purified DHFR confirmed that, in vitro, the protein was a monomer in the absence of dimerizer drug; in the presence of bisMTX, a complex of twice the monomeric molecular weight was observed. Furthermore, the off-rate was found to be 0.0002 s(-1), approximately 100 times slower than that reported for DHFR-MTX. Interestingly, the addition of excess bisMTX did not result in formation of the binary complex (1 protein:1 dimerizer) over the ternary complex (2 proteins:1 dimerizer), which suggests cooperative binding interactions (affinity modulation) between the two DHFR molecules in the bisMTX:DHFR(2) ternary complex. CONCLUSIONS: The combination of DHFR and bisMTX provides a new CID system with properties that could be useful for applications in vivo. Formation of the bisMTX:DHFR(2) ternary complex in vitro is promoted over a wide range of dimerizer concentrations, consistent with the idea that formation of the ternary complex recruits energetically favorable interactions between the DHFR monomers in the complex.
Asunto(s)
Metotrexato/química , Metotrexato/metabolismo , Tetrahidrofolato Deshidrogenasa/química , Tetrahidrofolato Deshidrogenasa/metabolismo , Sitios de Unión , Disponibilidad Biológica , Cromatografía en Gel , Dimerización , Escherichia coli/enzimología , Ligandos , Sustancias Macromoleculares , Metotrexato/análogos & derivados , Compuestos Organofosforados/químicaRESUMEN
Two activites causing nitrite disappearance are found in extracts of Neurospora; one, inducible by nitrate or nitrite and present only in nitrite-utilizing strains, catalyze the stoichiometric reduction of nitrite to ammonia; the other, present in all strains under all conditions, causes the disappearance of nitrite to something other than ammonia. The latter activity has a molecular weight of about 600 and may contain an oligopeptide, a metal, and an SH group(s). It has no known physiological function.
