Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 8 de 8
Filtrar
1.
PLoS Pathog ; 19(8): e1011532, 2023 08.
Artigo em Inglês | MEDLINE | ID: mdl-37531329

RESUMO

The COVID-19 pandemic represents a global challenge that has impacted and is expected to continue to impact the lives and health of people across the world for the foreseeable future. The rollout of vaccines has provided highly anticipated relief, but effective therapeutics are required to further reduce the risk and severity of infections. Monoclonal antibodies have been shown to be effective as therapeutics for SARS-CoV-2, but as new variants of concern (VoC) continue to emerge, their utility and use have waned due to limited or no efficacy against these variants. Furthermore, cumbersome systemic administration limits easy and broad access to such drugs. As well, concentrations of systemically administered antibodies in the mucosal epithelium, a primary site of initial infection, are dependent on neonatal Fc receptor mediated transport and require high drug concentrations. To reduce the viral load more effectively in the lung, we developed an inhalable formulation of a SARS-CoV-2 neutralizing antibody binding to a conserved epitope on the Spike protein, ensuring pan-neutralizing properties. Administration of this antibody via a vibrating mesh nebulization device retained antibody integrity and resulted in effective distribution of the antibody in the upper and lower respiratory tract of non-human primates (NHP). In comparison with intravenous administration, significantly higher antibody concentrations can be obtained in the lung, resulting in highly effective reduction in viral load post SARS-CoV-2 challenge. This approach may reduce the barriers of access and uptake of antibody therapeutics in real-world clinical settings and provide a more effective blueprint for targeting existing and potentially emerging respiratory tract viruses.


Assuntos
Antivirais , COVID-19 , Animais , Humanos , SARS-CoV-2 , Pandemias , Anticorpos Antivirais , Anticorpos Neutralizantes , Epitopos , Glicoproteína da Espícula de Coronavírus
2.
Nucleic Acids Res ; 42(16): 10711-9, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-25143529

RESUMO

The polymerization of amino acids into proteins occurs on ribosomes, with the rate influenced by the amino acids being polymerized. The imino acid proline is a poor donor and acceptor for peptide-bond formation, such that translational stalling occurs when three or more consecutive prolines (PPP) are encountered by the ribosome. In bacteria, stalling at PPP motifs is rescued by the elongation factor P (EF-P). Using SILAC mass spectrometry of Escherichia coli strains, we identified a subset of PPP-containing proteins for which the expression patterns remained unchanged or even appeared up-regulated in the absence of EF-P. Subsequent analysis using in vitro and in vivo reporter assays revealed that stalling at PPP motifs is influenced by the sequence context upstream of the stall site. Specifically, the presence of amino acids such as Cys and Thr preceding the stall site suppressed stalling at PPP motifs, whereas amino acids like Arg and His promoted stalling. In addition to providing fundamental insight into the mechanism of peptide-bond formation, our findings suggest how the sequence context of polyproline-containing proteins can be modulated to maximize the efficiency and yield of protein production.


Assuntos
Proteínas de Escherichia coli/química , Peptídeos , Biossíntese de Proteínas , Motivos de Aminoácidos , Sequência de Aminoácidos , Aminoácidos/análise , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas de Escherichia coli/biossíntese , Proteínas de Escherichia coli/genética , Peptídeos/análise , Ribossomos/metabolismo , Regulação para Cima
3.
Proc Natl Acad Sci U S A ; 110(38): 15265-70, 2013 Sep 17.
Artigo em Inglês | MEDLINE | ID: mdl-24003132

RESUMO

Ribosomes are the protein synthesizing factories of the cell, polymerizing polypeptide chains from their constituent amino acids. However, distinct combinations of amino acids, such as polyproline stretches, cannot be efficiently polymerized by ribosomes, leading to translational stalling. The stalled ribosomes are rescued by the translational elongation factor P (EF-P), which by stimulating peptide-bond formation allows translation to resume. Using metabolic stable isotope labeling and mass spectrometry, we demonstrate in vivo that EF-P is important for expression of not only polyproline-containing proteins, but also for specific subsets of proteins containing diprolyl motifs (XPP/PPX). Together with a systematic in vitro and in vivo analysis, we provide a distinct hierarchy of stalling triplets, ranging from strong stallers, such as PPP, DPP, and PPN to weak stallers, such as CPP, PPR, and PPH, all of which are substrates for EF-P. These findings provide mechanistic insight into how the characteristics of the specific amino acid substrates influence the fundamentals of peptide bond formation.


Assuntos
Escherichia coli K12/fisiologia , Fatores de Alongamento de Peptídeos/metabolismo , Prolina/metabolismo , Biossíntese de Proteínas/fisiologia , Ribossomos/metabolismo , Motivos de Aminoácidos/genética , Cromatografia Líquida , Escherichia coli K12/metabolismo , Humanos , Proteômica , Espectrometria de Massas em Tandem , beta-Galactosidase
4.
Nat Chem Biol ; 8(8): 695-7, 2012 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-22706199

RESUMO

Lys34 of the conserved translation elongation factor P (EF-P) is post-translationally lysinylated by YjeK and YjeA--a modification that is critical for bacterial virulence. Here we show that the currently accepted Escherichia coli EF-P modification pathway is incomplete and lacks a final hydroxylation step mediated by YfcM, an enzyme distinct from deoxyhypusine hydroxylase that catalyzes the final maturation step of eukaryotic initiation factor 5A, the eukaryotic EF-P homolog.


Assuntos
Proteínas de Escherichia coli/metabolismo , Escherichia coli/metabolismo , Regulação Bacteriana da Expressão Gênica/fisiologia , Lisina/química , Oxigenases de Função Mista/metabolismo , Fatores de Alongamento de Peptídeos/metabolismo , Quimotripsina/química , Escherichia coli/genética , Proteínas de Escherichia coli/genética , Espectrometria de Massas , Oxigenases de Função Mista/genética , Estrutura Molecular , Fatores de Alongamento de Peptídeos/genética , Ligação Proteica , Processamento de Proteína Pós-Traducional
5.
J Bacteriol ; 195(18): 4202-9, 2013 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-23852868

RESUMO

In all domains of life, initiator tRNA functions exclusively at the first step of protein synthesis while elongator tRNAs extend the polypeptide chain. Unique features of initiator tRNA enable it to preferentially bind the ribosomal P site and initiate translation. Recently, we showed that the abundance of initiator tRNA also contributes to its specialized role. This motivates the question, can a cell also use elongator tRNA to initiate translation under certain conditions? To address this, we introduced non-AUG initiation codons CCC (Pro), GAG (Glu), GGU (Gly), UCU (Ser), UGU (Cys), ACG (Thr), AAU (Asn), and AGA (Arg) into the uracil DNA glycosylase gene (ung) used as a reporter gene. Enzyme assays from log-phase cells revealed initiation from non-AUG codons when intracellular initiator tRNA levels were reduced. The activity increased significantly in stationary phase. Further increases in initiation from non-AUG codons occurred in both growth phases upon introduction of plasmid-borne genes of cognate elongator tRNAs. Since purine-rich Shine-Dalgarno sequences occur frequently on mRNAs (in places other than the canonical AUG codon initiation contexts), initiation with elongator tRNAs from the alternate contexts may generate proteome diversity under stress without compromising genomic integrity. Thus, by changing the relative amounts of initiator and elongator tRNAs within the cell, we have blurred the distinction between the two classes of tRNAs thought to be frozen through years of evolution.


Assuntos
Escherichia coli K12/genética , Escherichia coli K12/metabolismo , Elongação Traducional da Cadeia Peptídica/genética , Iniciação Traducional da Cadeia Peptídica/genética , RNA de Transferência/genética , Anticódon/metabolismo , Clonagem Molecular , Códon de Iniciação/química , Códon de Iniciação/metabolismo , Escherichia coli K12/crescimento & desenvolvimento , Immunoblotting , Elongação Traducional da Cadeia Peptídica/fisiologia , Iniciação Traducional da Cadeia Peptídica/fisiologia , Plasmídeos/genética , Biossíntese de Proteínas , RNA de Transferência/metabolismo , RNA de Transferência de Metionina/genética , RNA de Transferência de Metionina/metabolismo , Ribossomos/metabolismo
6.
FEBS J ; 275(15): 3772-82, 2008 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-18565105

RESUMO

Ribosome subunit assembly in bacteria is a fast and efficient process. Among the nonribosomal proteins involved in ribosome biogenesis are RNA helicases. We describe ribosome biogenesis in Escherichia coli strains lacking RNA helicase DeaD (CsdA) or DbpA. Ribosome large subunit assembly intermediate particles (40S) accumulate at 25 degrees C and at 37 degrees C in the absence of DeaD but not without DbpA. 23S rRNA is incompletely processed in the 40S and 50S particles of the DeaD(-) strain. Pulse labeling showed that the 40S particles are converted nearly completely into functional ribosomes. The rate of large ribosomal subunit assembly was reduced about four times in DeaD-deficient cells. Functional activity tests of the ribosomal particles demonstrated that the final step of 50S assembly, the activation step, was affected when DeaD was not present. The results are compatible with the model that predicts multiple DeaD-catalyzed structural transitions of the ribosome large subunit assembly.


Assuntos
RNA Helicases DEAD-box/metabolismo , Proteínas de Escherichia coli/metabolismo , Escherichia coli/enzimologia , Ribossomos/metabolismo , Sequência de Bases , RNA Helicases DEAD-box/genética , Primers do DNA , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas de Escherichia coli/genética , Cinética , RNA Ribossômico 23S/genética
7.
Cell Rep ; 9(2): 476-83, 2014 Oct 23.
Artigo em Inglês | MEDLINE | ID: mdl-25310979

RESUMO

Bacterial ribosomes stall on polyproline stretches and require the elongation factor P (EF-P) to relieve the arrest. Yet it remains unclear why evolution has favored the development of EF-P rather than selecting against the occurrence of polyproline stretches in proteins. We have discovered that only a single polyproline stretch is invariant across all domains of life, namely a proline triplet in ValS, the tRNA synthetase, that charges tRNA(Val) with valine. Here, we show that expression of ValS in vivo and in vitro requires EF-P and demonstrate that the proline triplet located in the active site of ValS is important for efficient charging of tRNA(Val) with valine and preventing formation of mischarged Thr-tRNA(Val) as well as efficient growth of E. coli in vivo. We suggest that the critical role of the proline triplet for ValS activity may explain why bacterial cells coevolved the EF-P rescue system.


Assuntos
Sequência Conservada , Proteínas de Escherichia coli/genética , Escherichia coli/genética , Fatores de Alongamento de Peptídeos/genética , Peptídeos/genética , Valina-tRNA Ligase/genética , Sequência de Aminoácidos , Escherichia coli/química , Escherichia coli/metabolismo , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/metabolismo , Evolução Molecular , Dados de Sequência Molecular , Mutação , Fatores de Alongamento de Peptídeos/química , Fatores de Alongamento de Peptídeos/metabolismo , Valina-tRNA Ligase/química , Valina-tRNA Ligase/metabolismo
8.
J Biol Chem ; 277(27): 24128-34, 2002 Jul 05.
Artigo em Inglês | MEDLINE | ID: mdl-11967262

RESUMO

The universally conserved 3'-terminal CCA sequence of tRNA interacts with large ribosomal subunit RNA during translation. The functional importance of the interaction between the 3'-terminal nucleotide of tRNA and the ribosome was studied in vitro using mutant in vitro transcribed tRNA(Val) A76G. Val-tRNA(CCG) does not support polypeptide synthesis on poly(GUA) as a message. However, in a co-translation system, where Val-tRNA(CCG) represented only a small fraction of total Val-tRNA, the mutant tRNA is able to transfer valine into a polypeptide chain, albeit at a reduced level. The A76G mutation does not affect binding of Val- or NAcVal-tRNA(CCG) to the A- or P-sites as shown by efficient peptide bond formation, although the donor activity of the mutant NAcVal-tRNA(CCG) in the peptidyl transfer reaction is slightly reduced compared with wild-type NAcVal-tRNA. Translocation of 3'-CCG-tRNA from the P- to the E-site is not significantly influenced. However, the A76G mutation drastically inhibits translocation of peptidyl-tRNA G(76) from the ribosomal A-site to the P-site, which apparently explains its failure to support cell-free protein synthesis. Our results indicate that the identity of the 3'-terminal nucleotide of tRNA is critical for tRNA movement in the ribosome.


Assuntos
Biossíntese de Proteínas , RNA de Transferência de Valina/genética , RNA de Transferência/química , Sequência de Bases , Variação Genética , Cinética , Mutagênese , Mutação Puntual , Poli G/biossíntese , Especificidade por Substrato
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA