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1.
G3 (Bethesda) ; 10(9): 3399-3402, 2020 09 02.
Artigo em Inglês | MEDLINE | ID: mdl-32763951

RESUMO

The world is facing a global pandemic of COVID-19 caused by the SARS-CoV-2 coronavirus. Here we describe a collection of codon-optimized coding sequences for SARS-CoV-2 cloned into Gateway-compatible entry vectors, which enable rapid transfer into a variety of expression and tagging vectors. The collection is freely available. We hope that widespread availability of this SARS-CoV-2 resource will enable many subsequent molecular studies to better understand the viral life cycle and how to block it.


Assuntos
Betacoronavirus/genética , Fases de Leitura Aberta/genética , Betacoronavirus/isolamento & purificação , COVID-19 , Clonagem Molecular , Infecções por Coronavirus/patologia , Infecções por Coronavirus/virologia , Escherichia coli/metabolismo , Humanos , Pandemias , Plasmídeos/genética , Plasmídeos/metabolismo , Pneumonia Viral/patologia , Pneumonia Viral/virologia , Potyvirus/genética , SARS-CoV-2
2.
Nat Biotechnol ; 36(1): 103-112, 2018 01.
Artigo em Inglês | MEDLINE | ID: mdl-29176613

RESUMO

Bacterial cell envelope protein (CEP) complexes mediate a range of processes, including membrane assembly, antibiotic resistance and metabolic coordination. However, only limited characterization of relevant macromolecules has been reported to date. Here we present a proteomic survey of 1,347 CEPs encompassing 90% inner- and outer-membrane and periplasmic proteins of Escherichia coli. After extraction with non-denaturing detergents, we affinity-purified 785 endogenously tagged CEPs and identified stably associated polypeptides by precision mass spectrometry. The resulting high-quality physical interaction network, comprising 77% of targeted CEPs, revealed many previously uncharacterized heteromeric complexes. We found that the secretion of autotransporters requires translocation and the assembly module TamB to nucleate proper folding from periplasm to cell surface through a cooperative mechanism involving the ß-barrel assembly machinery. We also establish that an ABC transporter of unknown function, YadH, together with the Mla system preserves outer membrane lipid asymmetry. This E. coli CEP 'interactome' provides insights into the functional landscape governing CE systems essential to bacterial growth, metabolism and drug resistance.


Assuntos
Membrana Celular/genética , Escherichia coli/genética , Complexos Multiproteicos/genética , Proteômica , Membrana Celular/química , Proteínas de Membrana/química , Proteínas de Membrana/classificação , Proteínas de Membrana/genética , Complexos Multiproteicos/química , Complexos Multiproteicos/classificação
3.
Environ Sci Technol ; 50(20): 11329-11336, 2016 Oct 18.
Artigo em Inglês | MEDLINE | ID: mdl-27682841

RESUMO

Determination of the physical interactions of environmental chemicals with cellular proteins is important for characterizing biological and toxic mechanism of action. Yet despite the discovery of numerous bioactive natural brominated compounds, such as hydroxylated polybrominated diphenyl ethers (OH-PBDEs), their corresponding protein targets remain largely unclear. Here, we reported a systematic and unbiased chemical proteomics assay (Target Identification by Ligand Stabilization, TILS) for target identification of bioactive molecules based on monitoring ligand-induced thermal stabilization. We first validated the broad applicability of this approach by identifying both known and unexpected proteins bound by diverse compounds (anticancer drugs, antibiotics). We then applied TILS to identify the bacterial target of 6-OH-BDE-47 as enoyl-acyl carrier protein reductase (FabI), an essential and widely conserved enzyme. Using affinity pull-down and in vitro enzymatic assays, we confirmed the potent antibacterial activity of 6-OH-BDE-47 occurs via direct binding and inhibition of FabI. Conversely, overexpression of FabI rescued the growth inhibition of Escherichia coli by 6-OH-BDE-47, validating it as the primary in vivo target. This study documents a chemical proteomics strategy for identifying the physical and functional targets of small molecules, and its potential high-throughput application to investigate the modes-of-action of environmental compounds.

4.
J Cell Biol ; 198(4): 623-36, 2012 Aug 20.
Artigo em Inglês | MEDLINE | ID: mdl-22908312

RESUMO

Systematic affinity purification combined with mass spectrometry analysis of N- and C-tagged cytoplasmic Hsp70/Hsp110 chaperones was used to identify new roles of Hsp70/Hsp110 in the cell. This allowed the mapping of a chaperone-protein network consisting of 1,227 unique interactions between the 9 chaperones and 473 proteins and highlighted roles for Hsp70/Hsp110 in 14 broad biological processes. Using this information, we uncovered an essential role for Hsp110 in spindle assembly and, more specifically, in modulating the activity of the widely conserved kinesin-5 motor Cin8. The role of Hsp110 Sse1 as a nucleotide exchange factor for the Hsp70 chaperones Ssa1/Ssa2 was found to be required for maintaining the proper distribution of kinesin-5 motors within the spindle, which was subsequently required for bipolar spindle assembly in S phase. These data suggest a model whereby the Hsp70-Hsp110 chaperone complex antagonizes Cin8 plus-end motility and prevents premature spindle elongation in S phase.


Assuntos
Proteínas de Choque Térmico HSP110/fisiologia , Proteínas de Choque Térmico HSP70/metabolismo , Fase S/fisiologia , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Fuso Acromático/fisiologia , Adenosina Trifosfatases/genética , Adenosina Trifosfatases/metabolismo , Células Cultivadas , Fatores de Troca do Nucleotídeo Guanina/fisiologia , Proteínas de Choque Térmico HSP70/genética , Cinesina/metabolismo , Cinesina/fisiologia , Proteínas Motores Moleculares/metabolismo , Saccharomyces cerevisiae/citologia , Proteínas de Saccharomyces cerevisiae/genética
5.
Mol Cell Proteomics ; 11(7): M111.016642, 2012 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-22357554

RESUMO

Bioactive molecules typically mediate their biological effects through direct physical association with one or more cellular proteins. The detection of drug-target interactions is therefore essential for the characterization of compound mechanism of action and off-target effects, but generic label-free approaches for detecting binding events in biological mixtures have remained elusive. Here, we report a method termed target identification by chromatographic co-elution (TICC) for routinely monitoring the interaction of drugs with cellular proteins under nearly physiological conditions in vitro based on simple liquid chromatographic separations of cell-free lysates. Correlative proteomic analysis of drug-bound protein fractions by shotgun sequencing is then performed to identify candidate target(s). The method is highly reproducible, does not require immobilization or derivatization of drug or protein, and is applicable to diverse natural products and synthetic compounds. The capability of TICC to detect known drug-protein target physical interactions (K(d) range: micromolar to nanomolar) is demonstrated both qualitatively and quantitatively. We subsequently used TICC to uncover the sterol biosynthetic enzyme Erg6p as a novel putative anti-fungal target. Furthermore, TICC identified Asc1 and Dak1, a core 40 S ribosomal protein that represses gene expression, and dihydroxyacetone kinase involved in stress adaptation, respectively, as novel yeast targets of a dopamine receptor agonist.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Antifúngicos/metabolismo , Proteínas Fúngicas/metabolismo , Proteínas de Ligação ao GTP/metabolismo , Metiltransferases/metabolismo , Fosfotransferases (Aceptor do Grupo Álcool)/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/análise , Sistema Livre de Células , Cromatografia Líquida , Agonistas de Dopamina/metabolismo , Escherichia coli , Proteínas Fúngicas/análise , Proteínas de Ligação ao GTP/análise , Células HeLa , Humanos , Ácidos Hidroxâmicos/metabolismo , Indenos/metabolismo , Macrolídeos/metabolismo , Espectrometria de Massas , Metotrexato/metabolismo , Metiltransferases/análise , Terapia de Alvo Molecular , Fosfotransferases (Aceptor do Grupo Álcool)/análise , Ligação Proteica , Proteômica , Saccharomyces cerevisiae , Proteínas de Saccharomyces cerevisiae/análise
6.
PLoS Genet ; 7(11): e1002377, 2011 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-22125496

RESUMO

As the interface between a microbe and its environment, the bacterial cell envelope has broad biological and clinical significance. While numerous biosynthesis genes and pathways have been identified and studied in isolation, how these intersect functionally to ensure envelope integrity during adaptive responses to environmental challenge remains unclear. To this end, we performed high-density synthetic genetic screens to generate quantitative functional association maps encompassing virtually the entire cell envelope biosynthetic machinery of Escherichia coli under both auxotrophic (rich medium) and prototrophic (minimal medium) culture conditions. The differential patterns of genetic interactions detected among > 235,000 digenic mutant combinations tested reveal unexpected condition-specific functional crosstalk and genetic backup mechanisms that ensure stress-resistant envelope assembly and maintenance. These networks also provide insights into the global systems connectivity and dynamic functional reorganization of a universal bacterial structure that is both broadly conserved among eubacteria (including pathogens) and an important target.


Assuntos
Membrana Celular/genética , Epistasia Genética/genética , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas de Membrana/genética , Proteínas Associadas aos Microtúbulos/genética , Meios de Cultura , Resistência a Medicamentos/genética , Escherichia coli/crescimento & desenvolvimento , Regulação Bacteriana da Expressão Gênica , Interação Gene-Ambiente , Proteínas de Membrana/metabolismo , Redes e Vias Metabólicas/genética , Microscopia Eletrônica , Proteínas Associadas aos Microtúbulos/metabolismo , Anotação de Sequência Molecular , Análise de Sequência com Séries de Oligonucleotídeos
7.
Mol Microbiol ; 79(2): 484-502, 2011 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-21219465

RESUMO

Clustered Regularly Interspaced Short Palindromic Repeats (CRISPRs) and the associated proteins (Cas) comprise a system of adaptive immunity against viruses and plasmids in prokaryotes. Cas1 is a CRISPR-associated protein that is common to all CRISPR-containing prokaryotes but its function remains obscure. Here we show that the purified Cas1 protein of Escherichia coli (YgbT) exhibits nuclease activity against single-stranded and branched DNAs including Holliday junctions, replication forks and 5'-flaps. The crystal structure of YgbT and site-directed mutagenesis have revealed the potential active site. Genome-wide screens show that YgbT physically and genetically interacts with key components of DNA repair systems, including recB, recC and ruvB. Consistent with these findings, the ygbT deletion strain showed increased sensitivity to DNA damage and impaired chromosomal segregation. Similar phenotypes were observed in strains with deletion of CRISPR clusters, suggesting that the function of YgbT in repair involves interaction with the CRISPRs. These results show that YgbT belongs to a novel, structurally distinct family of nucleases acting on branched DNAs and suggest that, in addition to antiviral immunity, at least some components of the CRISPR-Cas system have a function in DNA repair.


Assuntos
Colífagos/crescimento & desenvolvimento , Enzimas Reparadoras do DNA/metabolismo , Reparo do DNA , Endodesoxirribonucleases/metabolismo , Proteínas de Escherichia coli/metabolismo , Escherichia coli/enzimologia , Escherichia coli/virologia , Sequências Repetitivas de Ácido Nucleico , Proteínas Associadas a CRISPR , Cristalografia por Raios X , Enzimas Reparadoras do DNA/química , Enzimas Reparadoras do DNA/genética , Desoxirribonucleases/química , Desoxirribonucleases/genética , Desoxirribonucleases/metabolismo , Endodesoxirribonucleases/química , Endodesoxirribonucleases/genética , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/genética , Deleção de Genes , Modelos Moleculares , Mutagênese Sítio-Dirigida , Proteínas Mutantes/genética , Proteínas Mutantes/metabolismo
8.
Mol Cell Proteomics ; 9(11): 2460-73, 2010 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-20467045

RESUMO

Effective methods to detect and quantify functionally linked regulatory proteins in complex biological samples are essential for investigating mammalian signaling pathways. Traditional immunoassays depend on proprietary reagents that are difficult to generate and multiplex, whereas global proteomic profiling can be tedious and can miss low abundance proteins. Here, we report a target-driven liquid chromatography-tandem mass spectrometry (LC-MS/MS) strategy for selectively examining the levels of multiple low abundance components of signaling pathways which are refractory to standard shotgun screening procedures and hence appear limited in current MS/MS repositories. Our stepwise approach consists of: (i) synthesizing microscale peptide arrays, including heavy isotope-labeled internal standards, for use as high quality references to (ii) build empirically validated high density LC-MS/MS detection assays with a retention time scheduling system that can be used to (iii) identify and quantify endogenous low abundance protein targets in complex biological mixtures with high accuracy by correlation to a spectral database using new software tools. The method offers a flexible, rapid, and cost-effective means for routine proteomic exploration of biological systems including "label-free" quantification, while minimizing spurious interferences. As proof-of-concept, we have examined the abundance of transcription factors and protein kinases mediating pluripotency and self-renewal in embryonic stem cell populations.


Assuntos
Cromatografia Líquida/métodos , Peptídeos/química , Análise Serial de Proteínas/métodos , Proteínas/análise , Espectrometria de Massas em Tandem/métodos , Sequência de Aminoácidos , Animais , Camundongos , Peptídeos/síntese química , Peptídeos/genética , Proteômica/métodos
9.
Methods Mol Biol ; 564: 373-400, 2009.
Artigo em Inglês | MEDLINE | ID: mdl-19544035

RESUMO

Biochemical purification of affinity-tagged proteins in combination with mass spectrometry methods is increasingly seen as a cornerstone of systems biology, as it allows for the systematic genome-scale characterization of macromolecular protein complexes, representing demarcated sets of stably interacting protein partners. Accurate and sensitive identification of both the specific and shared polypeptide components of distinct complexes requires purification to near homogeneity. To this end, a sequential peptide affinity (SPA) purification system was developed to enable the rapid and efficient isolation of native Escherichia coli protein complexes (J Proteome Res 3:463-468, 2004). SPA purification makes use of a dual-affinity tag, consisting of three modified FLAG sequences (3X FLAG) and a calmodulin binding peptide (CBP), spaced by a cleavage site for tobacco etch virus (TEV) protease (J Proteome Res 3:463-468, 2004). Using the lambda-phage Red homologous recombination system (PNAS 97:5978-5983, 2000), a DNA cassette, encoding the SPA-tag and a selectable marker flanked by gene-specific targeting sequences, is introduced into a selected locus in the E. coli chromosome so as to create a C-terminal fusion with the protein of interest. This procedure aims for near-endogenous levels of tagged protein production in the recombinant bacteria to avoid spurious, non-specific protein associations (J Proteome Res 3:463-468, 2004). In this chapter, we describe a detailed, optimized protocol for the tagging, purification, and subsequent mass spectrometry-based identification of the subunits of even low-abundance bacterial protein complexes isolated as part of an ongoing large-scale proteomic study in E. coli (Nature 433:531-537, 2005).


Assuntos
Cromatografia de Afinidade/métodos , Proteínas de Escherichia coli/metabolismo , Escherichia coli/metabolismo , Complexos Multiproteicos/isolamento & purificação , Proteômica/métodos , Espectrometria de Massas em Tandem/métodos , Sequência de Aminoácidos , Sequência de Bases , Western Blotting , Cromatografia Líquida/métodos , Eletroforese em Gel Bidimensional , Endopeptidases/genética , Escherichia coli/genética , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/isolamento & purificação , Medições Luminescentes , Dados de Sequência Molecular , Complexos Multiproteicos/química , Fragmentos de Peptídeos/análise , Fragmentos de Peptídeos/química , Mapeamento de Peptídeos , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/isolamento & purificação
10.
PLoS Biol ; 7(4): e96, 2009 Apr 28.
Artigo em Inglês | MEDLINE | ID: mdl-19402753

RESUMO

One-third of the 4,225 protein-coding genes of Escherichia coli K-12 remain functionally unannotated (orphans). Many map to distant clades such as Archaea, suggesting involvement in basic prokaryotic traits, whereas others appear restricted to E. coli, including pathogenic strains. To elucidate the orphans' biological roles, we performed an extensive proteomic survey using affinity-tagged E. coli strains and generated comprehensive genomic context inferences to derive a high-confidence compendium for virtually the entire proteome consisting of 5,993 putative physical interactions and 74,776 putative functional associations, most of which are novel. Clustering of the respective probabilistic networks revealed putative orphan membership in discrete multiprotein complexes and functional modules together with annotated gene products, whereas a machine-learning strategy based on network integration implicated the orphans in specific biological processes. We provide additional experimental evidence supporting orphan participation in protein synthesis, amino acid metabolism, biofilm formation, motility, and assembly of the bacterial cell envelope. This resource provides a "systems-wide" functional blueprint of a model microbe, with insights into the biological and evolutionary significance of previously uncharacterized proteins.


Assuntos
Proteínas de Escherichia coli/genética , Escherichia coli/genética , Genoma Bacteriano , Proteoma/genética , Escherichia coli/metabolismo , Proteínas de Escherichia coli/metabolismo , Complexos Multiproteicos/genética , Mapeamento de Interação de Proteínas/métodos
11.
Nat Methods ; 5(9): 789-95, 2008 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-18677321

RESUMO

Physical and functional interactions define the molecular organization of the cell. Genetic interactions, or epistasis, tend to occur between gene products involved in parallel pathways or interlinked biological processes. High-throughput experimental systems to examine genetic interactions on a genome-wide scale have been devised for Saccharomyces cerevisiae, Schizosaccharomyces pombe, Caenorhabditis elegans and Drosophila melanogaster, but have not been reported previously for prokaryotes. Here we describe the development of a quantitative screening procedure for monitoring bacterial genetic interactions based on conjugation of Escherichia coli deletion or hypomorphic strains to create double mutants on a genome-wide scale. The patterns of synthetic sickness and synthetic lethality (aggravating genetic interactions) we observed for certain double mutant combinations provided information about functional relationships and redundancy between pathways and enabled us to group bacterial gene products into functional modules.


Assuntos
Escherichia coli/genética , Análise de Sequência com Séries de Oligonucleotídeos , Conjugação Genética , Genoma Bacteriano , Mutação
12.
Cell ; 131(5): 915-26, 2007 Nov 30.
Artigo em Inglês | MEDLINE | ID: mdl-18045534

RESUMO

In response to genotoxic stress, cells evoke a plethora of physiological responses collectively aimed at enhancing viability and maintaining the integrity of the genome. Here, we report that unspliced tRNA rapidly accumulates in the nuclei of yeast Saccharomyces cerevisiae after DNA damage. This response requires an intact MEC1- and RAD53-dependent signaling pathway that impedes the nuclear export of intron-containing tRNA via differential relocalization of the karyopherin Los1 to the cytoplasm. The accumulation of unspliced tRNA in the nucleus signals the activation of Gcn4 transcription factor, which, in turn, contributes to cell-cycle arrest in G1 in part by delaying accumulation of the cyclin Cln2. The regulated nucleocytoplasmic tRNA trafficking thus constitutes an integral physiological adaptation to DNA damage. These data further illustrate how signal-mediated crosstalk between distinct functional modules, namely, tRNA nucleocytoplasmic trafficking, protein synthesis, and checkpoint execution, allows for functional coupling of tRNA biogenesis and cell-cycle progression.


Assuntos
Transporte Ativo do Núcleo Celular/fisiologia , Ciclo Celular/fisiologia , Dano ao DNA/fisiologia , RNA de Transferência/metabolismo , Saccharomyces cerevisiae/genética , Processamento Alternativo/fisiologia , Fatores de Transcrição de Zíper de Leucina Básica , Ciclo Celular/genética , Proteínas de Ciclo Celular/genética , Sobrevivência Celular/genética , Quinase do Ponto de Checagem 2 , Ciclinas/metabolismo , Proteínas de Ligação a DNA/metabolismo , Regulação para Baixo , Deleção de Genes , Genes cdc , Modelos Biológicos , Complexo de Proteínas Formadoras de Poros Nucleares/genética , Organismos Geneticamente Modificados , Proteínas Serina-Treonina Quinases/genética , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Transdução de Sinais/genética , Fatores de Transcrição/metabolismo
13.
BMC Biol ; 5: 38, 2007 Sep 19.
Artigo em Inglês | MEDLINE | ID: mdl-17880717

RESUMO

BACKGROUND: Histone modifications have been implicated in the regulation of transcription and, more recently, in DNA replication and repair. In yeast, a major conserved histone acetyltransferase, Hat1p, preferentially acetylates lysine residues 5 and 12 on histone H4. RESULTS: Here, we report that a nuclear sub-complex consisting of Hat1p and its partner Hat2p interacts physically and functionally with the origin recognition complex (ORC). While mutational inactivation of the histone acetyltransferase (HAT) gene HAT1 alone does not compromise origin firing or initiation of DNA replication, a deletion in HAT1 (or HAT2) exacerbates the growth defects of conditional orc-ts mutants. Thus, the ORC-associated Hat1p-dependent histone acetyltransferase activity suggests a novel linkage between histone modification and DNA replication. Additional genetic and biochemical evidence points to the existence of partly overlapping histone H3 acetyltransferase activities in addition to Hat1p/Hat2p for proper DNA replication efficiency. Furthermore, we demonstrated a dynamic association of Hat1p with chromatin during S-phase that suggests a role of this enzyme at the replication fork. CONCLUSION: We have found an intriguing new association of the Hat1p-dependent histone acetyltransferase in addition to its previously known role in nuclear chromatin assembly (Hat1p/Hat2p-Hif1p). The participation of a distinct Hat1p/Hat2p sub-complex suggests a linkage of histone H4 modification with ORC-dependent DNA replication.


Assuntos
Acetiltransferases/metabolismo , Replicação do DNA , Histonas/metabolismo , Complexo de Reconhecimento de Origem/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/enzimologia , Acetilação , Acetiltransferases/genética , Alelos , Western Blotting , Imunoprecipitação da Cromatina , DNA Fúngico/genética , DNA Fúngico/metabolismo , Regulação Fúngica da Expressão Gênica , Genes Fúngicos , Histona Acetiltransferases , Histonas/genética , Lisina/metabolismo , Mutação , Fases de Leitura Aberta , Complexo de Reconhecimento de Origem/genética , Fase S , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética
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