RESUMEN

Deposition of amyloid-beta peptide (Abeta) in the brain is an early and invariant feature of all forms of Alzheimer's disease (AD). As for all proteins or peptides, the steady-state level of Abeta peptide is determined not only by its production, but also by its degradation. So, overactive proteases involved in generating Abeta from amyloid precursor protein or underactive Abeta-degrading enzymes could lead to abnormal Abeta deposition in the brain. Since in the sporadic forms of AD (90% of all AD cases) an impaired clearance of Abeta appears to be at the origin of its aggregation and tissue deposition, we have investigated its proteolytic degradation by several neuronal and non-neuronal cells. In this report, we show that these cell types exhibit a similar profile of Abeta-degradation by cell-surface and secreted proteases which were respectively characterized as metallo- and serine proteases. By using a combination of the liquid chromatography/on-line mass spectrometry, we demonstrate that: (i)-the membrane associated protease(s) hydrolizes Abeta40 essentially at Lys(28) Gly(29), Phe(19) Phe(20) and Val(18) Phe(19) bonds; and (ii)-the secreted protease(s) cleaves the generating fragments Abeta (1-28), Abeta (1-19), Abeta (1-18) at His(14) Gln(15) bond and also Abeta (1-28) at Phe(20) Ala(21) and Asp(23) Val(24) sites. This is the first time our results define a proteolytic degradation process of Abeta40 that appears to be independent of the cell type and may represent a general pattern of its enzymatic clearance.

Asunto(s)

Péptidos beta-Amiloides/metabolismo , Neuronas/metabolismo , Fragmentos de Péptidos/metabolismo , Péptido Hidrolasas/metabolismo , Secuencia de Aminoácidos , Péptidos beta-Amiloides/química , Línea Celular , Membrana Celular/enzimología , Supervivencia Celular , Cromatografía Líquida de Alta Presión , Medios de Cultivo , Ensayo de Inmunoadsorción Enzimática , Espacio Extracelular/metabolismo , Espacio Intracelular/metabolismo , Espectrometría de Masas , Datos de Secuencia Molecular , Neuronas/química , Neuronas/enzimología , Neuropéptido Y/metabolismo , Fragmentos de Péptidos/química , Espectrofotometría Ultravioleta

RESUMEN

D-aspartate ligase has remained the last unidentified peptide bond-forming enzyme in the peptidoglycan assembly pathway of Gram-positive bacteria. Here we show that a two-gene cluster of Enterococcus faecium encodes aspartate racemase (Racfm) and ligase (Aslfm) for incorporation of D-Asp into the side chain of the peptidoglycan precursor. Aslfm was identified as a new member of the ATP-grasp protein superfamily, which includes a diverse set of enzymes catalyzing ATP-dependent carboxylate-amine ligation reactions. Aslfm specifically ligated the beta-carboxylate of D-Asp to the epsilon-amino group of L-Lys in the nucleotide precursor UDP-N-acetylmuramyl-pentapeptide. D-iso-asparagine was not a substrate of Aslfm, indicating that the presence of this amino acid in the peptidoglycan of E. faecium results from amidation of the alpha-carboxyl of D-Asp after its addition to the precursor. Heterospecific expression of the genes encoding Racfm and Aslfm in Enterococcus faecalis led to production of stem peptides substituted by D-Asp instead of L-Ala2, providing evidence for the in vivo specificity and function of these enzymes. Strikingly, sequencing of the cross-bridges revealed that substitution of L-Ala2 by D-Asp is tolerated by the d,d-transpeptidase activity of the penicillin-binding proteins both in the acceptor and in the donor substrates. The Aslfm ligase appears as an attractive target for the development of narrow spectrum antibiotics active against multiresistant E. faecium.

Asunto(s)

Ácido D-Aspártico/metabolismo , Enterococcus faecium/enzimología , Ligasas/metabolismo , Peptidoglicano/biosíntesis , Adenosina Trifosfato/metabolismo , Isomerasas de Aminoácido/metabolismo , Secuencia de Aminoácidos , Sustitución de Aminoácidos , Ligasas/aislamiento & purificación , Datos de Secuencia Molecular , Peptidoglicano/química , Homología de Secuencia de Aminoácido , Especificidad por Sustrato

RESUMEN

The beta-lactam antibiotics remain the most commonly used to treat severe infections. Because of structural similarity between the beta-lactam ring and the d-alanyl(4)-d-alanine(5) extremity of bacterial cell wall precursors, the drugs act as suicide substrates of the dd-transpeptidases that catalyze the last cross-linking step of cell wall assembly. Here, we show that this mechanism of action can be defeated by a novel type of transpeptidase identified for the first time by reverse genetics in abeta-lactam-resistant mutant of Enterococcus faecium. The enzyme, Ldt(fm), catalyzes in vitro the cross-linking of peptidoglycan subunits in a beta-lactam-insensitive ld-transpeptidation reaction. The specificity of Ldt(fm) for the l-lysyl(3)-d-alanine(4) peptide bond of tetrapeptide donors accounts for resistance because the substrate does not mimic beta-lactams in contrast to d-alanyl(4)-d-alanine(5) in the pentapeptide donors required for dd-transpeptidation. Ldt(fm) homologues are encountered sporadically among taxonomically distant bacteria, indicating that ld-transpeptidase-mediated resistance may emerge in various pathogens.

Asunto(s)

Enterococcus faecium/enzimología , Peptidoglicano/química , Alanina/química , Secuencia de Aminoácidos , Ampicilina/farmacología , Animales , Antibacterianos/farmacología , Encéfalo/microbiología , Catálisis , Pared Celular/metabolismo , Cromatografía , Cromatografía por Intercambio Iónico , Reactivos de Enlaces Cruzados/farmacología , Relación Dosis-Respuesta a Droga , Farmacorresistencia Bacteriana , Enterococcus faecium/metabolismo , Escherichia coli/metabolismo , Espectrometría de Masas , Modelos Biológicos , Modelos Químicos , Modelos Genéticos , Datos de Secuencia Molecular , Péptidos/química , Peptidil Transferasas/química , Unión Proteica , Especificidad por Sustrato , beta-Lactamas/química , beta-Lactamas/farmacología

RESUMEN

Bioassay-guided fractionation of cytotoxic of methanol extract of the seeds of Annona cherimola provided two novel cyclic peptides, cherimolacyclopeptide E (1) and cherimolacyclopeptide F (2), which exhibited significant cytotoxic activity against the KB (human nasopharyngeal carcinoma) cell culture system. The peptide 1 and 2 were elucidated by MS/MS fragmentation experiments using a Q-TOF mass spectrometer equipped with an ESI source, extensive 2D NMR analyses and chemical degradation.

Asunto(s)

Annona/química , Antineoplásicos Fitogénicos/aislamiento & purificación , Péptidos Cíclicos/aislamiento & purificación , Secuencia de Aminoácidos , Antineoplásicos Fitogénicos/química , Antineoplásicos Fitogénicos/farmacología , Supervivencia Celular/efectos de los fármacos , Ensayos de Selección de Medicamentos Antitumorales , Cromatografía de Gases y Espectrometría de Masas , Humanos , Concentración 50 Inhibidora , Células KB , Espectroscopía de Resonancia Magnética , Datos de Secuencia Molecular , Péptidos Cíclicos/química , Péptidos Cíclicos/farmacología , Semillas/química , Espectrometría de Masa por Ionización de Electrospray

RESUMEN

The seeds of Annona glauca furnished two cyclopeptides one of which is novel. The structure was elucidated on the basis on mass spectrometry, 2D NMR methods and amino acids analysis.

Asunto(s)

Annona/química , Péptidos Cíclicos/química , Semillas/química , Antineoplásicos Fitogénicos/química , Antineoplásicos Fitogénicos/farmacología , Humanos , Células KB , Estructura Molecular , Péptidos Cíclicos/farmacología

RESUMEN

Four new ether diglycosides (1-4), named matayosides A-D, were isolated from the root bark of Matayba guianensis, a plant exhibiting in vitro antiplasmodial activity. They were identified as hexadecyl-[O-2,3,4-tri-O-acetyl-alpha-L-rhamnopyranosyl-(1-->2)]-6-O-palmitoyl-beta-D-glucopyranoside, hexadecyl-[O-2,3,4-tri-O-acetyl-alpha-L-rhamnopyranosyl-(1-->2)]-4,6-di-O-acetyl-beta-D-glucopyranoside, hexadecyl-[O-2,3,4-tri-O-acetyl-alpha-L-rhamnopyranosyl-(1-->2)]-3,6-di-O-acetyl-beta-D-glucopyranoside and hexadecyl-[O-2,3,4-tri-O-acetyl-alpha-L-rhamnopyranosyl-(1-->2)]-6-O-acetyl-beta-D-glucopyranoside, respectively. Their structures were established using one- and two-dimensional NMR techniques, mass spectrometry (MS) and MS/MS experiments. The compounds were found to inhibit the growth of Plasmodium falciparum in vitro with IC50 values ranging from 2.5 to 8.9 microg/mL.

Asunto(s)

Antimaláricos/aislamiento & purificación , Antimaláricos/farmacología , Glicósidos/aislamiento & purificación , Glicósidos/farmacología , Sapindaceae/química , Animales , Conformación de Carbohidratos , Éteres , Plasmodium falciparum/efectos de los fármacos , Análisis Espectral

RESUMEN

In a chemical investigation of the seeds of Annona cherimola, a natural cyclic heptapeptide, cherimolacyclopeptide D, were isolated and purified by HPLC with three known cyclic peptides, cherimolacyclopeptides A, B and C. The structure was established by various analyses including MS/MS fragmentation, spectroscopic and chemical evidences.

Asunto(s)

Annona/química , Péptidos Cíclicos/aislamiento & purificación , Semillas/química , Estructura Molecular , Péptidos Cíclicos/química

RESUMEN

From a methanol extract of the seeds of Annona cherimola, a new cyclic heptapeptide, cherimolacyclopeptide C, has been isolated. The structure was elucidated on the basis of the MS/MS fragmentation using a Q-TOF mass spectrometer equipped with an ESI source, extensive 2D NMR experiments, and chemical degradation. Cherimolacyclopeptide C exhibited significant in vitro cytotoxic activity against KB cells, with an IC(50) value of 0.072 microM.

Asunto(s)

Annona/química , Antineoplásicos Fitogénicos/aislamiento & purificación , Péptidos Cíclicos/aislamiento & purificación , Plantas Medicinales/química , Secuencia de Aminoácidos , Antineoplásicos Fitogénicos/química , Antineoplásicos Fitogénicos/farmacología , Ensayos de Selección de Medicamentos Antitumorales , Humanos , Concentración 50 Inhibidora , Células KB , Estructura Molecular , Péptidos Cíclicos/química , Péptidos Cíclicos/farmacología , Semillas/química , España

RESUMEN

The peptidoglycan cross-bridges of Staphylococcus aureus, Enterococcus faecalis, and Enterococcus faecium consist of the sequences Gly(5), l-Ala(2), and d-Asx, respectively. Expression of the fmhB, femA, and femB genes of S. aureus in E. faecalis led to the production of peptidoglycan precursors substituted by mosaic side chains that were efficiently used by the penicillin-binding proteins for cross-bridge formation. The Fem transferases were specific for incorporation of glycyl residues at defined positions of the side chains in the absence of any additional S. aureus factors such as tRNAs used for amino acid activation. The PBPs of E. faecalis displayed a broad substrate specificity because mosaic side chains containing from 1 to 5 residues and Gly instead of l-Ala at the N-terminal position were used for peptidoglycan cross-linking. Low affinity PBP2a of S. aureus conferred beta-lactam resistance in E. faecalis and E. faecium, thereby indicating that there was no barrier to heterospecific expression of resistance caused by variations in the structure of peptidoglycan precursors. Thus, conservation of the structure of the peptidoglycan cross-bridges in members of the same species reflects the high specificity of the enzymes for side chain synthesis, although this is not essential for the activity of the PBPs.

Asunto(s)

Bacterias Grampositivas/metabolismo , Peptidoglicano/biosíntesis , Secuencia de Aminoácidos , Enterococcus faecalis/metabolismo , Enterococcus faecium/metabolismo , Peptidoglicano/química , Peptidil Transferasas/metabolismo , Staphylococcus aureus/metabolismo , Especificidad por Sustrato

RESUMEN

Peptidoglycan polymerization complexes contain multimodular penicillin-binding proteins (PBP) of classes A and B that associate a conserved C-terminal transpeptidase module to an N-terminal glycosyltransferase or morphogenesis module, respectively. In Enterococcus faecalis, class B PBP5 mediates intrinsic resistance to the cephalosporin class of beta-lactam antibiotics, such as ceftriaxone. To identify the glycosyltransferase partner(s) of PBP5, combinations of deletions were introduced in all three class A PBP genes of E. faecalis JH2-2 (ponA, pbpF, and pbpZ). Among mutants with single or double deletions, only JH2-2 DeltaponA DeltapbpF was susceptible to ceftriaxone. Ceftriaxone resistance was restored by heterologous expression of pbpF from Enterococcus faecium but not by mgt encoding the monofunctional glycosyltransferase of Staphylococcus aureus. Thus, PBP5 partners essential for peptidoglycan polymerization in the presence of beta-lactams formed a subset of the class A PBPs of E. faecalis, and heterospecific complementation was observed with an ortholog from E. faecium. Site-directed mutagenesis of pbpF confirmed that the catalytic serine residue of the transpeptidase module was not required for resistance. None of the three class A PBP genes was essential for viability, although deletion of the three genes led to an increase in the generation time and to a decrease in peptidoglycan cross-linking. As the E. faecalis chromosome does not contain any additional glycosyltransferase-related genes, these observations indicate that glycan chain polymerization in the triple mutant is performed by a novel type of glycosyltransferase. The latter enzyme was not inhibited by moenomycin, since deletion of the three class A PBP genes led to high-level resistance to this glycosyltransferase inhibitor.

Asunto(s)

Proteínas Bacterianas/metabolismo , Proteínas Portadoras/metabolismo , Enterococcus faecalis/efectos de los fármacos , Hexosiltransferasas/metabolismo , Muramoilpentapéptido Carboxipeptidasa/metabolismo , Peptidil Transferasas/metabolismo , Resistencia betalactámica , Proteínas Bacterianas/clasificación , Proteínas Bacterianas/genética , Proteínas Portadoras/clasificación , Proteínas Portadoras/genética , Enterococcus faecalis/genética , Enterococcus faecalis/crecimiento & desarrollo , Inhibidores Enzimáticos/farmacología , Eliminación de Gen , Glicosiltransferasas/antagonistas & inhibidores , Hexosiltransferasas/clasificación , Hexosiltransferasas/genética , Pruebas de Sensibilidad Microbiana , Muramoilpentapéptido Carboxipeptidasa/clasificación , Muramoilpentapéptido Carboxipeptidasa/genética , Oligosacáridos/farmacología , Proteínas de Unión a las Penicilinas , Peptidoglicano/química , Peptidoglicano/metabolismo , Peptidil Transferasas/clasificación , Peptidil Transferasas/genética

RESUMEN

Enterococcus faecalis produces a specific penicillin-binding protein (PBP5) that mediates high-level resistance to the cephalosporin class of beta-lactam antibiotics. Deletion of a locus encoding a previously uncharacterized two-component regulatory system of E. faecalis (croRS) led to a 4,000-fold reduction in the MIC of the expanded-spectrum cephalosporin ceftriaxone. The cytoplasmic domain of the sensor kinase (CroS) was purified and shown to catalyze ATP-dependent autophosphorylation followed by transfer of the phosphate to the mated response regulator (CroR). The croR and croS genes were cotranscribed from a promoter (croRp) located in the rrnC-croR intergenic region. A putative seryl-tRNA synthetase gene (serS) located immediately downstream from croS did not appear to be a target of CroRS regulation or to play a role in ceftriaxone resistance. A plasmid-borne croRp-lacZ fusion was trans-activated by the CroRS system in response to the presence of ceftriaxone in the culture medium. The fusion was also induced by representatives of other classes of beta-lactam antibiotics and by inhibitors of early and late steps of peptidoglycan synthesis. The croRS null mutant produced PBP5, and expression of an additional copy of pbp5 under the control of a heterologous promoter did not restore ceftriaxone resistance. Deletion of croRS was not associated with any defect in the synthesis of the nucleotide precursor UDP-MurNAc-pentapeptide or of the D-Ala(4)-->L-Ala-L-Ala-Lys(3) peptidoglycan cross-bridge. Thus, the croRS mutant was susceptible to ceftriaxone despite the production of PBP5 and the synthesis of wild-type peptidoglycan precursors. These observations constitute the first description of regulatory genes essential for PBP5-mediated beta-lactam resistance in enterococci.

Asunto(s)

Enterococcus faecalis/efectos de los fármacos , Enterococcus faecalis/genética , Resistencia betalactámica/fisiología , Antibacterianos/farmacología , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Secuencia de Carbohidratos , Ceftriaxona/farmacología , Mapeo Cromosómico , Citoplasma/metabolismo , Enterococcus faecalis/metabolismo , Eliminación de Gen , Datos de Secuencia Molecular , Peptidoglicano/metabolismo , Regiones Promotoras Genéticas , ARN Mensajero