Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 8 de 8
Filtrar
1.
Bioorg Med Chem Lett ; 23(6): 1727-31, 2013 Mar 15.
Artigo em Inglês | MEDLINE | ID: mdl-23414806

RESUMO

A novel series of 3-O-carbamoyl erythromycin A derived analogs, labeled carbamolides, with activity versus resistant bacterial isolates of staphylococci (including macrolide and oxazolidinone resistant strains) and streptococci are reported. An (R)-2-aryl substituent on a pyrrolidine carbamate appeared to be critical for achieving potency against resistant strains. Crystal structures showed a distinct aromatic interaction between the (R)-2-aryl (3-pyridyl for 4d) substituent on the pyrrolidine and G2484 (G2505, Escherichia coli) of the Deinococcus radiodurans 50S ribosome (3.2Å resolution).


Assuntos
Antibacterianos/química , Eritromicina/análogos & derivados , Compostos de Metilureia/química , Staphylococcus/isolamento & purificação , Streptococcus/isolamento & purificação , Antibacterianos/síntese química , Sítios de Ligação , Cristalografia por Raios X , Deinococcus/metabolismo , Farmacorresistência Bacteriana , Eritromicina/síntese química , Escherichia coli/metabolismo , Testes de Sensibilidade Microbiana , Estrutura Terciária de Proteína , Pirrolidinas/química , Subunidades Ribossômicas Maiores de Bactérias/química , Subunidades Ribossômicas Maiores de Bactérias/metabolismo , Staphylococcus/efeitos dos fármacos , Streptococcus/efeitos dos fármacos
2.
RNA ; 14(1): 188-95, 2008 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-17998293

RESUMO

We have developed a novel chromatography for the rapid isolation of active ribosomes from bacteria without the use of harsh conditions or lengthy procedures that damage ribosomes. Ribosomes interact with an alkyl linker attached to the resin, apparently through their RNA component. Examples are given with ribosomes from Escherichia coli, Deinococcus radiodurans, and with clinical isolates of Streptococcus pneumoniae and methicillin-resistant Staphylococcus aureus (MRSA). The ribosomes obtained by this method are unusually intact, so that highly active ribosomes can now be isolated from the clinical isolates, enabling significantly improved in vitro functional assays that will greatly assist the discovery and development of new ribosomally targeted antibiotics.


Assuntos
Bactérias/ultraestrutura , Cromatografia Líquida/métodos , Ribossomos , Bactérias/patogenicidade , Espectrometria de Massas , RNA Bacteriano/química , RNA Bacteriano/isolamento & purificação
3.
Acta Crystallogr D Biol Crystallogr ; 65(Pt 12): 1270-82, 2009 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-19966413

RESUMO

A systematic analysis was undertaken to seek correlations between the integrity, purity and activity of 50S ribosomal subunit preparations from Deinococcus radiodurans and their ability to crystallize. Conditions of fermentation, purification and crystallization were varied in a search for crystals that could reliably supply an industrial X-ray crystallography program for the structure-based design of ribosomal antibiotics. A robust protocol was obtained to routinely obtain crystals that gave diffraction patterns extending to 2.9 A resolution and that were large enough to yield a complete data set from a single crystal. To our knowledge, this is the most systematic study of this challenging area so far undertaken. Ribosome crystallization is a complex multi-factorial problem and although a clear correlation of crystallization with subunit properties was not obtained, the search for key factors that potentiate crystallization has been greatly narrowed and promising areas for further inquiry are suggested.


Assuntos
Proteínas de Bactérias/química , Deinococcus/química , Proteínas Ribossômicas/química , Subunidades Ribossômicas Maiores de Bactérias/química , Sequência de Aminoácidos , Proteínas de Bactérias/isolamento & purificação , Fracionamento Celular , Cristalografia por Raios X , Deinococcus/genética , Deinococcus/crescimento & desenvolvimento , Modelos Moleculares , Dados de Sequência Molecular , Conformação de Ácido Nucleico , Estrutura Quaternária de Proteína , RNA Bacteriano/química , RNA Bacteriano/genética , RNA Bacteriano/isolamento & purificação , Proteínas Ribossômicas/isolamento & purificação , Subunidades Ribossômicas Maiores de Bactérias/genética
4.
Anal Biochem ; 395(1): 77-85, 2009 Dec 01.
Artigo em Inglês | MEDLINE | ID: mdl-19646947

RESUMO

We have developed an affinity purification of the large ribosomal subunit from Deinococcus radiodurans that exploits its association with FLAG-tagged 30S subunits. Thus, capture is indirect so that no modification of the 50S is required and elution is achieved under mild conditions (low magnesium) that disrupt the association, avoiding the addition of competitor ligands or coelution of common contaminants. Efficient purification of highly pure 50S is achieved, and the chromatography simultaneously sorts the 50S into three classes according to their association status (unassociated, loosely associated, or tightly associated), improving homogeneity.


Assuntos
Deinococcus/ultraestrutura , Subunidades Ribossômicas Maiores de Bactérias/química , Proteínas de Bactérias/análise , Centrifugação com Gradiente de Concentração , Cromatografia de Afinidade , Cromatografia Líquida de Alta Pressão , Clonagem Molecular , Bases de Dados de Proteínas , Expressão Gênica , Cloreto de Magnésio , Oligopeptídeos , Fragmentos de Peptídeos/análise , Peptídeos/genética , RNA Bacteriano/análise , RNA Ribossômico/análise , Proteínas Recombinantes de Fusão , Proteínas Ribossômicas/análise , Proteínas Ribossômicas/genética , Subunidades Ribossômicas Maiores de Bactérias/metabolismo , Subunidades Ribossômicas Menores de Bactérias/genética , Subunidades Ribossômicas Menores de Bactérias/metabolismo , Espectrometria de Massas por Ionização por Electrospray , Espectrometria de Massas em Tandem
5.
Cell Chem Biol ; 23(11): 1362-1371, 2016 Nov 17.
Artigo em Inglês | MEDLINE | ID: mdl-27746128

RESUMO

Proprotein convertase subtilisin/kexin type 9 (PCSK9) is a secreted protein that downregulates low-density lipoprotein (LDL) receptor (LDL-R) levels on the surface of hepatocytes, resulting in decreased clearance of LDL-cholesterol (LDL-C). Phenotypic screening of a small-molecule compound collection was used to identify an inhibitor of PCSK9 secretion, (R)-N-(isoquinolin-1-yl)-3-(4-methoxyphenyl)-N-(piperidin-3-yl)propanamide (R-IMPP), which was shown to stimulate uptake of LDL-C in hepatoma cells by increasing LDL-R levels, without altering levels of secreted transferrin. Systematic investigation of the mode of action revealed that R-IMPP did not decrease PCSK9 transcription or increase PCSK9 degradation, but instead caused transcript-dependent inhibition of PCSK9 translation. In support of this surprising mechanism of action, we found that R-IMPP was able to selectively bind to human, but not E. coli, ribosomes. This study opens a new avenue for the development of drugs that modulate the activity of target proteins by mechanisms involving inhibition of eukaryotic translation.


Assuntos
Isoquinolinas/farmacologia , Inibidores de PCSK9 , Pró-Proteína Convertase 9/metabolismo , Biossíntese de Proteínas/efeitos dos fármacos , Ribossomos/efeitos dos fármacos , Bibliotecas de Moléculas Pequenas/farmacologia , Linhagem Celular Tumoral , Humanos , Isoquinolinas/química , Ribossomos/metabolismo , Bibliotecas de Moléculas Pequenas/química
6.
Cold Spring Harb Protoc ; 2015(4): 359-62, 2015 Apr 01.
Artigo em Inglês | MEDLINE | ID: mdl-25834264

RESUMO

Mixed-mode chromatography on cysteine-SulfoLink resin efficiently separates ribosomes from cell lysates and is particularly effective at rapidly removing endogenous proteases and nucleases, resulting in ribosomes of improved purity, integrity, and activity. Binding occurs partly by anion exchange of the RNA of the ribosomes, so that cells must be lysed in a buffer of moderate ionic strength (conductivity no more than 20 mS for chromatography of bacterial ribosomes) without any highly charged additives (e.g., heparin, which is used to inhibit RNases in yeast). A robust protocol for Escherichia coli is given here as an example.


Assuntos
Cromatografia/métodos , Escherichia coli/metabolismo , Ribossomos/metabolismo , Ribonucleases/metabolismo
7.
Microbiol Mol Biol Rev ; 73(1): 22-35, 2009 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-19258531

RESUMO

The assembly of bacterial ribosomes is viewed with increasing interest as a potential target for new antibiotics. The in vivo synthesis and assembly of ribosomes are briefly reviewed here, highlighting the many ways in which assembly can be perturbed. The process is compared with the model in vitro process from which much of our knowledge is derived. The coordinate synthesis of the ribosomal components is essential for their ordered and efficient assembly; antibiotics interfere with this coordination and therefore affect assembly. It has also been claimed that the binding of antibiotics to nascent ribosomes prevents their assembly. These two contrasting models of antibiotic action are compared and evaluated. Finally, the suitability and tractability of assembly as a drug target are assessed.


Assuntos
Antibacterianos/farmacologia , Bactérias/efeitos dos fármacos , Bactérias/metabolismo , Ribossomos/efeitos dos fármacos , Ribossomos/metabolismo , Antibacterianos/metabolismo , Proteínas de Bactérias/metabolismo , Inibidores da Síntese de Proteínas/farmacologia , RNA Ribossômico/metabolismo , Proteínas Ribossômicas/metabolismo
8.
Mol Cell ; 20(3): 427-35, 2005 Nov 11.
Artigo em Inglês | MEDLINE | ID: mdl-16285924

RESUMO

Deletion of the gene for protein L27 from the E. coli chromosome results in severe defects in cell growth. This deficiency is corrected by the expression of wild-type (wt) protein L27 from a plasmid. Examination of strains expressing L27 variants truncated at the N terminus reveals that the absence of as few as three amino acids leads to a decrease in growth rate, an impairment in peptidyl transferase activity, and a sharp decline in the labeling of L27 from the 3' end of a photoreactive tRNA at the ribosomal P site. These findings suggest that the flexible N-terminal sequence of L27, which protrudes onto the interface of the bacterial 50S subunit, can reach the peptidyl transferase active site and contribute to its function, possibly by helping to correctly position tRNA substrates at the catalytic site.


Assuntos
Cromossomos Bacterianos , Proteínas de Escherichia coli/metabolismo , Escherichia coli/metabolismo , Deleção de Genes , Proteínas Ribossômicas/metabolismo , Ribossomos/metabolismo , Sítios de Ligação/fisiologia , Cromossomos Bacterianos/genética , Cromossomos Bacterianos/metabolismo , Escherichia coli/genética , Proteínas de Escherichia coli/genética , Peptidil Transferases/genética , Peptidil Transferases/metabolismo , Biossíntese de Proteínas/fisiologia , RNA Bacteriano/genética , RNA Bacteriano/metabolismo , RNA de Transferência/genética , RNA de Transferência/metabolismo , Proteínas Ribossômicas/genética , Ribossomos/genética
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA