RESUMEN
The solution structure of filipin III, an antifungal polyene macrolide biosynthesized by Streptomyces filipinensis and widely used for the detection and the quantitation of cholesterol in biomembranes, has been calculated with a set of geometrical restraints derived from 1H NMR in DMSO-d(6) at 25 degrees C. Filipin III appears as a rod-shaped molecule of 18 A length. Its amphiphilic structure is made of an all-syn 1,3-polyol motif, stabilized by intramolecular hydrogen bonds on one side, and a conjugated pentaene moiety on the other side of the molecule. The overall shape is comparable to cholesterol, and the molecular structure of filipin III affords a first molecular basis to the comprehensive understanding of the interactions possible in the filipin III-cholesterol complex which is still unknown at the atomic resolution.
Asunto(s)
Antifúngicos/química , Filipina/química , Resonancia Magnética Nuclear Biomolecular , Streptomyces/química , Antifúngicos/metabolismo , Colesterol/química , Colesterol/metabolismo , Filipina/metabolismo , Enlace de Hidrógeno , Modelos Moleculares , Estructura Secundaria de Proteína , SolucionesRESUMEN
Plipastatins A and B are antifungal antibiotics belonging to a family of lipopeptides capable of inhibiting phospholipase A(2) (PLA(2)) and are biosynthesised under certain circumstances by Bacillus subtilis. U-(15)N plipastatins A and B were obtained from cultures of the strain NCIB 8872 on a Landy medium modified for stable-isotope labelling by the substitution of the L-glutamic acid used as the sole nitrogen source, by (15)NH(4)Cl. These two lipo-decapeptides, lactonised by esterification of the Ile10 C-terminus with the phenolic hydroxyl of Tyr3, differ only by a D-Ala (plipastatin A)/D-Val (plipastatin B) substitution at the position 6. The (1)H- and (15)N-nuclear magnetic resonance (NMR) signals of a 4:6 mixture of plipastatins A and B were unambiguously assigned and their structures in dimethylsulfoxide solution were calculated on the basis of a set of NMR-derived restraints. Plipastatins A and B are well-defined structures in solution stabilised by a type 1 beta-turn comprising residues 6-9 and several other specific hydrogen bonds. The structures afford a first molecular basis for the future studies of their biological activities both in lipidic layers or on PLA(2).
Asunto(s)
Antifúngicos/química , Bacillus subtilis/metabolismo , Inhibidores Enzimáticos/química , Ácidos Grasos/química , Espectroscopía de Resonancia Magnética , Oligopéptidos/química , Fosfolipasas A/antagonistas & inhibidores , Secuencia de Aminoácidos , Cloruro de Amonio/metabolismo , Esterificación , Ácidos Grasos/biosíntesis , Enlace de Hidrógeno , Marcaje Isotópico , Estructura Molecular , Isótopos de Nitrógeno , Oligopéptidos/biosíntesis , Péptidos Cíclicos , Homología de Secuencia , SolucionesRESUMEN
The eukaryotic translation initiation factor eIF4E is a potent oncogene elevated in many cancers, including the M4 and M5 subtypes of acute myeloid leukemia (AML). Although eIF4E RNA levels are elevated 3- to 10-fold in M4/M5 AML, the molecular underpinnings of this dysregulation were unknown. Here, we demonstrate that EIF4E is a direct transcriptional target of nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) that is dysregulated preferentially in M4 and M5 AML. In primary hematopoietic cells and in cell lines, eIF4E levels are induced by NF-κB activating stimuli. Pharmacological or genetic inhibition of NF-κB represses this activation. The endogenous human EIF4E promoter recruits p65 and cRel to evolutionarily conserved κB sites in vitro and in vivo following NF-κB activation. Transcriptional activation is demonstrated by recruitment of p300 to the κB sites and phosphorylated Pol II to the coding region. In primary AML specimens, generally we observe that substantially more NF-κB complexes associate with eIF4E promoter elements in M4 and M5 AML specimens examined than in other subtypes or unstimulated normal primary hematopoietic cells. Consistently, genetic inhibition of NF-κB abrogates eIF4E RNA levels in this same population. These findings provide novel insights into the transcriptional control of eIF4E and a novel molecular basis for its dysregulation in at least a subset of M4/M5 AML specimens.
Asunto(s)
Factor 4E Eucariótico de Iniciación/genética , Regulación Leucémica de la Expresión Génica , Leucemia Mieloide Aguda/genética , FN-kappa B/metabolismo , Linfocitos B/citología , Linfocitos B/metabolismo , Estudios de Casos y Controles , Núcleo Celular/genética , Células Cultivadas , Factor 4E Eucariótico de Iniciación/metabolismo , Humanos , Leucemia Mieloide Aguda/clasificación , Leucemia Mieloide Aguda/patología , FN-kappa B/genética , Regiones Promotoras Genéticas/genética , Activación TranscripcionalRESUMEN
The antifungal antibiotic lipopeptide bacillomycin L [cyclo-(L-Asp1-D-Tyr2-D-Asn3-L-Ser4-L-Gln5-D-Ser6++ +-L-Thr7-beta-amino fatty acid)] from Bacillus subtilis belongs to the iturinic family of antifungal agents and acts with a strict sterol-phospholipid dependence on biomembranes. This antibiotic has been analysed using solution NMR spectroscopy in its native active form and its inactive (L-Asp1, D-Tyr2) di-O-methylated form. The structures were calculated under NMR-derived restraints using molecular-dynamic simulated-annealing protocols starting from a random array of atoms. The structure of the native antibiotic is spread over different conformers in which two families are recognized. It was found that most structures have dihedral phi and psi angles defining a type-II' beta-turn including amino acids 5-8, in certain cases stabilized by a 8HN-5CO hydrogen bond, whereas a minority of structures adopt an inverse gamma-turn including amino acids 6-8, stabilized in all cases by an 8HN-6CO hydrogen bond. The di-O-methylation of L-Asp1 and D-Tyr2, an amino acid strictly conserved within the iturinic group of antibiotics, does not induce major differences in the NMR spectra and in the NMR structures. The results are discussed in relation to the specific loss of interaction with sterols when the native antifungal bacillomycin L is methylated on the conserved D-Tyr2 position.
Asunto(s)
Antifúngicos/química , Esteroles/química , Secuencia de Aminoácidos , Enlace de Hidrógeno , Espectroscopía de Resonancia Magnética , Modelos Moleculares , Péptidos Cíclicos/química , Conformación ProteicaRESUMEN
Previous NMR studies on surfactin proposed two gamma or beta-turn-containing conformers while recent CD studies described beta-sheets and alpha-helices in surfactin. Since these data were not obtained in the same conditions, the conformation of surfactin was reinvestigated by FTIR spectroscopy, a diagnostic method for beta-sheets. In trifluoroethanol, the FTIR spectra of surfactin and its diester are compatible with gamma and/or beta-turn(s) and the differences in their CD spectra show the importance of the Glu(1) and Asp(5) COOH groups in stabilizing the lipopeptide conformation. The calcium-induced spectral changes of both lipopeptides suggest a first binding of the divalent ions to the surfactin COOH groups (until calcium-lipopeptide mole ratio reached 1) followed by bulk conformational changes (at higher mole ratios). In Tris buffer at pH 8.5, the FTIR amide I band shape, without the typical 1610-1628 and 1675-1695 cm(-1) bands, ascertains the absence of beta-sheets.