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1.
iScience ; 26(9): 107563, 2023 Sep 15.
Artigo em Inglês | MEDLINE | ID: mdl-37664601

RESUMO

In a scenario where the discovery of new molecules to fight antibiotic resistance is a public health concern, ribosomally synthesized and post-translationally modified peptides constitute a promising alternative. In this context, the Gram-positive human gut symbiont Ruminococcus gnavus E1 produces five sactipeptides, Ruminococcins C1 to C5 (RumC1-C5), co-expressed with two radical SAM maturases. RumC1 has been shown to be effective against various multidrug resistant Gram-positives clinical isolates. Here, after adapting the biosynthesis protocol to obtain the four mature RumC2-5 we then evaluate their antibacterial activities. Establishing first that both maturases exhibit substrate tolerance, we then observed a variation in the antibacterial efficacy between the five isoforms. We established that all RumCs are safe for humans with interesting multifunctionalities. While no synergies where observed for the five RumCs, we found a synergistic action with conventional antibiotics targeting the cell wall. Finally, we identified crucial residues for antibacterial activity of RumC isoforms.

2.
Int J Mol Sci ; 23(17)2022 Aug 31.
Artigo em Inglês | MEDLINE | ID: mdl-36077319

RESUMO

RNA polymerases (RNAPs) are found in all living organisms. In the chloroplasts, the plastid-encoded RNA polymerase (PEP) is a prokaryotic-type multimeric RNAP involved in the selective transcription of the plastid genome. One of its active states requires the assembly of nuclear-encoded PEP-Associated Proteins (PAPs) on the catalytic core, producing a complex of more than 900 kDa, regarded as essential for chloroplast biogenesis. In this study, sequence alignments of the catalytic core subunits across various chloroplasts of the green lineage and prokaryotes combined with structural data show that variations are observed at the surface of the core, whereas internal amino acids associated with the catalytic activity are conserved. A purification procedure compatible with a structural analysis was used to enrich the native PEP from Sinapis alba chloroplasts. A mass spectrometry (MS)-based proteomic analysis revealed the core components, the PAPs and additional proteins, such as FLN2 and pTAC18. MS coupled with crosslinking (XL-MS) provided the initial structural information in the form of protein clusters, highlighting the relative position of some subunits with the surfaces of their interactions. Using negative stain electron microscopy, the PEP three-dimensional envelope was calculated. Particles classification shows that the protrusions are very well-conserved, offering a framework for the future positioning of all the PAPs. Overall, the results show that PEP-associated proteins are firmly and specifically associated with the catalytic core, giving to the plastid transcriptional complex a singular structure compared to other RNAPs.


Assuntos
Proteínas de Arabidopsis , Sinapis , Proteínas de Arabidopsis/genética , Cloroplastos/genética , Cloroplastos/metabolismo , RNA Polimerases Dirigidas por DNA/genética , RNA Polimerases Dirigidas por DNA/metabolismo , Regulação da Expressão Gênica de Plantas , Plastídeos/genética , Plastídeos/metabolismo , Proteômica , Sinapis/metabolismo
3.
Nanoscale ; 13(19): 8901-8908, 2021 May 20.
Artigo em Inglês | MEDLINE | ID: mdl-33949561

RESUMO

Well-organized protein assemblies offer many properties that justify their use for the design of innovative bionanomaterials. Herein, crystals of the oligomerization domain of the LEAFY protein from Ginkgo biloba, organized in a honeycomb architecture, were used as a modular platform for the selective grafting of a ruthenium-based complex. The resulting bio-hybrid crystalline material was fully characterized by UV-visible and Raman spectroscopy and by mass spectrometry and LC-MS analysis after selective enzymatic digestion. Interestingly, insertion of complexes within the tubular structure affords an impressive increase in stability of the crystals, eluding the use of stabilizing cross-linking strategies.


Assuntos
Ginkgo biloba , Folhas de Planta , Cromatografia Líquida , Espectrometria de Massas , Proteínas
4.
Proteomes ; 9(2)2021 Apr 23.
Artigo em Inglês | MEDLINE | ID: mdl-33922761

RESUMO

(1) Background: The proteomic analysis of histones constitutes a delicate task due to the combination of two factors: slight variations in the amino acid sequences of variants and the multiplicity of post-translational modifications (PTMs), particularly those occurring on lysine residues. (2) Methods: To dissect the relationship between both aspects, we carefully evaluated PTM identification on lysine 27 from histone H3 (H3K27) and the artefactual chemical modifications that may lead to erroneous PTM determination. H3K27 is a particularly interesting example because it can bear a range of PTMs and it sits nearby residues 29 and 31 that vary between H3 sequence variants. We discuss how the retention times, neutral losses and immonium/diagnostic ions observed in the MS/MS spectra of peptides bearing modified lysines detectable in the low-mass region might help validate the identification of modified sequences. (3) Results: Diagnostic ions carry key information, thereby avoiding potential mis-identifications due to either isobaric PTM combinations or isobaric amino acid-PTM combinations. This also includes cases where chemical formylation or acetylation of peptide N-termini artefactually occurs during sample processing or simply in the timeframe of LC-MS/MS analysis. Finally, in the very subtle case of positional isomers possibly corresponding to a given mass of lysine modification, the immonium and diagnostic ions may allow the identification of the in vivo structure.

5.
Mol Plant Pathol ; 21(12): 1620-1633, 2020 12.
Artigo em Inglês | MEDLINE | ID: mdl-33029918

RESUMO

Pectin is synthesized in a highly methylesterified form in the Golgi cisternae and partially de-methylesterified in muro by pectin methylesterases (PMEs). Arabidopsis thaliana produces a local and strong induction of PME activity during the infection of the necrotrophic fungus Botrytis cinerea. AtPME17 is a putative A. thaliana PME highly induced in response to B. cinerea. Here, a fine tuning of AtPME17 expression by different defence hormones was identified. Our genetic evidence demonstrates that AtPME17 strongly contributes to the pathogen-induced PME activity and resistance against B. cinerea by triggering jasmonic acid-ethylene-dependent PDF1.2 expression. AtPME17 belongs to group 2 isoforms of PMEs characterized by a PME domain preceded by an N-terminal PRO region. However, the biochemical evidence for AtPME17 as a functional PME is still lacking and the role played by its PRO region is not known. Using the Pichia pastoris expression system, we demonstrate that AtPME17 is a functional PME with activity favoured by an increase in pH. AtPME17 performs a blockwise pattern of pectin de-methylesterification that favours the formation of egg-box structures between homogalacturonans. Recombinant AtPME17 expression in Escherichia coli reveals that the PRO region acts as an intramolecular inhibitor of AtPME17 activity.


Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/enzimologia , Botrytis/fisiologia , Hidrolases de Éster Carboxílico/metabolismo , Defensinas/metabolismo , Pectinas/metabolismo , Doenças das Plantas/imunologia , Arabidopsis/genética , Arabidopsis/imunologia , Arabidopsis/microbiologia , Proteínas de Arabidopsis/genética , Hidrolases de Éster Carboxílico/genética , Ciclopentanos/metabolismo , Defensinas/genética , Escherichia coli/genética , Escherichia coli/metabolismo , Etilenos/metabolismo , Expressão Gênica , Isoenzimas , Oxilipinas/metabolismo , Doenças das Plantas/microbiologia , Regiões Promotoras Genéticas/genética , Proteínas Recombinantes , Saccharomycetales/genética , Saccharomycetales/metabolismo
6.
Proc Natl Acad Sci U S A ; 117(32): 19168-19177, 2020 08 11.
Artigo em Inglês | MEDLINE | ID: mdl-32719135

RESUMO

The emergence of superbugs developing resistance to antibiotics and the resurgence of microbial infections have led scientists to start an antimicrobial arms race. In this context, we have previously identified an active RiPP, the Ruminococcin C1, naturally produced by Ruminococcus gnavus E1, a symbiont of the healthy human intestinal microbiota. This RiPP, subclassified as a sactipeptide, requires the host digestive system to become active against pathogenic Clostridia and multidrug-resistant strains. Here we report its unique compact structure on the basis of four intramolecular thioether bridges with reversed stereochemistry introduced posttranslationally by a specific radical-SAM sactisynthase. This structure confers to the Ruminococcin C1 important clinical properties including stability to digestive conditions and physicochemical treatments, a higher affinity for bacteria than simulated intestinal epithelium, a valuable activity at therapeutic doses on a range of clinical pathogens, mediated by energy resources disruption, and finally safety for human gut tissues.


Assuntos
Antibacterianos/química , Antibacterianos/farmacologia , Clostridiales/química , Peptídeos/química , Peptídeos/farmacologia , Antibacterianos/isolamento & purificação , Infecções Bacterianas/tratamento farmacológico , Infecções Bacterianas/microbiologia , Humanos , Mucosa Intestinal/efeitos dos fármacos , Mucosa Intestinal/microbiologia , Peptídeos/isolamento & purificação
7.
Nucleic Acids Res ; 48(8): 4115-4138, 2020 05 07.
Artigo em Inglês | MEDLINE | ID: mdl-32182340

RESUMO

Epigenetic regulation of gene expression is tightly controlled by the dynamic modification of histones by chemical groups, the diversity of which has largely expanded over the past decade with the discovery of lysine acylations, catalyzed from acyl-coenzymes A. We investigated the dynamics of lysine acetylation and crotonylation on histones H3 and H4 during mouse spermatogenesis. Lysine crotonylation appeared to be of significant abundance compared to acetylation, particularly on Lys27 of histone H3 (H3K27cr) that accumulates in sperm in a cleaved form of H3. We identified the genomic localization of H3K27cr and studied its effects on transcription compared to the classical active mark H3K27ac at promoters and distal enhancers. The presence of both marks was strongly associated with highest gene expression. Assessment of their co-localization with transcription regulators (SLY, SOX30) and chromatin-binding proteins (BRD4, BRDT, BORIS and CTCF) indicated systematic highest binding when both active marks were present and different selective binding when present alone at chromatin. H3K27cr and H3K27ac finally mark the building of some sperm super-enhancers. This integrated analysis of omics data provides an unprecedented level of understanding of gene expression regulation by H3K27cr in comparison to H3K27ac, and reveals both synergistic and specific actions of each histone modification.


Assuntos
Elementos Facilitadores Genéticos , Epigênese Genética , Código das Histonas , Regiões Promotoras Genéticas , Espermatogênese/genética , Acetilcoenzima A/metabolismo , Acetilação , Acil Coenzima A/metabolismo , Animais , Evolução Biológica , Crotonatos/metabolismo , Genômica , Histonas/química , Histonas/metabolismo , Lisina/metabolismo , Masculino , Metabolômica , Camundongos Endogâmicos C57BL , Proteômica , Transcrição Gênica , Leveduras/metabolismo , Leveduras/fisiologia
8.
Sci Adv ; 5(9): eaaw9969, 2019 09.
Artigo em Inglês | MEDLINE | ID: mdl-31579822

RESUMO

A major public health challenge today is the resurgence of microbial infections caused by multidrug-resistant strains. Consequently, novel antimicrobial molecules are actively sought for development. In this context, the human gut microbiome is an under-explored potential trove of valuable natural molecules, such as the ribosomally-synthesized and post-translationally modified peptides (RiPPs). The biological activity of the sactipeptide subclass of RiPPs remains under-characterized. Here, we characterize an antimicrobial sactipeptide, Ruminococcin C1, purified from the caecal contents of rats mono-associated with Ruminococcus gnavus E1, a human symbiont. Its heterologous expression and post-translational maturation involving a specific sactisynthase establish a thioether network, which creates a double-hairpin folding. This original structure confers activity against pathogenic Clostridia and multidrug-resistant strains but no toxicity towards eukaryotic cells. Therefore, the Ruminococcin C1 should be considered as a valuable candidate for drug development and its producer strain R. gnavus E1 as a relevant probiotic for gut health enhancement.


Assuntos
Antibiose , Microbioma Gastrointestinal , Ruminococcus/fisiologia , Simbiose , Sequência de Aminoácidos , Animais , Peptídeos Catiônicos Antimicrobianos/biossíntese , Peptídeos Catiônicos Antimicrobianos/química , Farmacorresistência Bacteriana Múltipla , Humanos , Proteólise , Ratos , Ruminococcus/efeitos dos fármacos
9.
Methods Mol Biol ; 2030: 1-10, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31347105

RESUMO

Recombinant proteins are essential components of therapeutic, biotechnological, food, and household products. In some cases, recombinant proteins must be purified and their quantity and/or concentration precisely determined. In this chapter, we describe a protocol for the quantification of purified recombinant proteins. The protocol is based on a microwave-assisted acidic hydrolysis of the target protein followed by high-resolution mass spectrometry (HRMS) analysis of the hydrolytic products. Absolute quantification is obtained by adding controlled amounts of labeled amino acids that serve as standards.


Assuntos
Aminoácidos/análise , Espectrometria de Massas/métodos , Proteínas Recombinantes/análise , Aminoácidos/química , Aminoácidos/efeitos da radiação , Cromatografia Líquida de Alta Pressão/métodos , Hidrólise/efeitos da radiação , Micro-Ondas , Proteínas Recombinantes/química , Proteínas Recombinantes/efeitos da radiação
10.
Nat Microbiol ; 4(7): 1208-1220, 2019 07.
Artigo em Inglês | MEDLINE | ID: mdl-31036909

RESUMO

The protozoan parasite Toxoplasma gondii has co-evolved with its homeothermic hosts (humans included) strategies that drive its quasi-asymptomatic persistence in hosts, hence optimizing the chance of transmission to new hosts. Persistence, which starts with a small subset of parasites that escape host immune killing and colonize the so-called immune privileged tissues where they differentiate into a low replicating stage, is driven by the interleukin 12 (IL-12)-interferon-γ (IFN-γ) axis. Recent characterization of a family of Toxoplasma effectors that are delivered into the host cell, in which they rewire the host cell gene expression, has allowed the identification of regulators of the IL-12-IFN-γ axis, including repressors. We now report on the dense granule-resident effector, called TEEGR (Toxoplasma E2F4-associated EZH2-inducing gene regulator) that counteracts the nuclear factor-κB (NF-κB) signalling pathway. Once exported into the host cell, TEEGR ends up in the nucleus where it not only complexes with the E2F3 and E2F4 host transcription factors to induce gene expression, but also promotes shaping of a non-permissive chromatin through its capacity to switch on EZH2. Remarkably, EZH2 fosters the epigenetic silencing of a subset of NF-κB-regulated cytokines, thereby strongly contributing to the host immune equilibrium that influences the host immune response and promotes parasite persistence in mice.


Assuntos
Proteína Potenciadora do Homólogo 2 de Zeste/metabolismo , NF-kappa B/metabolismo , Proteínas de Protozoários/metabolismo , Transdução de Sinais/genética , Toxoplasma/fisiologia , Animais , Linhagem Celular , Núcleo Celular/metabolismo , Citocinas/metabolismo , Fatores de Transcrição E2F/genética , Fatores de Transcrição E2F/metabolismo , Proteína Potenciadora do Homólogo 2 de Zeste/genética , Expressão Gênica , Regulação da Expressão Gênica , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Mutação , Carga Parasitária , Regiões Promotoras Genéticas , Multimerização Proteica , Proteínas de Protozoários/genética , Toxoplasma/genética , Toxoplasma/imunologia , Toxoplasmose/metabolismo , Toxoplasmose/parasitologia
11.
Proteomics ; 17(1-2)2017 01.
Artigo em Inglês | MEDLINE | ID: mdl-28008711

RESUMO

A proteomics assay was set up to analyze food substrates for eight toxins of the CBRN (chemical, biological, radiological and nuclear) threat, namely ricin, Clostridium perfringens epsilon toxin (ETX), Staphylococcus aureus enterotoxins (SEA, SEB and SED), shigatoxins from Shigella dysenteriae and entero-hemorragic Escherichia coli strains (STX1 and STX2) and Campylobacter jejuni cytolethal distending toxin (CDT). The assay developed was based on an antibody-free sample preparation followed by bottom-up LC-MS/MS analysis operated in targeted mode. Highly specific detection and absolute quantification were obtained using isotopically labeled proteins (PSAQ standards) spiked into the food matrix. The sensitivity of the assay for the eight toxins was lower than the oral LD50 which would likely be used in a criminal contamination of food supply. This assay should be useful in monitoring biological threats. In the public-health domain, it opens the way for multiplex investigation of food-borne toxins using targeted LC-MS/MS.


Assuntos
Proteômica/métodos , Toxinas Bacterianas/análise , Cromatografia Líquida , Enterotoxinas/análise , Toxina Shiga/análise , Espectrometria de Massas em Tandem
12.
PLoS Pathog ; 12(1): e1005361, 2016 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-26735307

RESUMO

Discoveries made over the past ten years have provided evidence that invertebrate antiparasitic responses may be primed in a sustainable manner, leading to the failure of a secondary encounter with the same pathogen. This phenomenon called "immune priming" or "innate immune memory" was mainly phenomenological. The demonstration of this process remains to be obtained and the underlying mechanisms remain to be discovered and exhaustively tested with rigorous functional and molecular methods, to eliminate all alternative explanations. In order to achieve this ambitious aim, the present study focuses on the Lophotrochozoan snail, Biomphalaria glabrata, in which innate immune memory was recently reported. We provide herein the first evidence that a shift from a cellular immune response (encapsulation) to a humoral immune response (biomphalysin) occurs during the development of innate memory. The molecular characterisation of this process in Biomphalaria/Schistosoma system was undertaken to reconcile mechanisms with phenomena, opening the way to a better comprehension of innate immune memory in invertebrates. This prompted us to revisit the artificial dichotomy between innate and memory immunity in invertebrate systems.


Assuntos
Biomphalaria/imunologia , Interações Hospedeiro-Parasita/imunologia , Imunidade Celular/imunologia , Imunidade Humoral/imunologia , Memória Imunológica/imunologia , Animais , Biomphalaria/parasitologia , Vetores de Doenças , Imunidade Inata/imunologia , RNA Interferente Pequeno , Reação em Cadeia da Polimerase em Tempo Real , Schistosoma mansoni/imunologia , Esquistossomose mansoni/imunologia , Esquistossomose mansoni/veterinária , Transfecção
13.
Environ Microbiol ; 17(4): 1152-65, 2015 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-24919412

RESUMO

Vibrio tasmaniensis LGP32, a facultative intracellular pathogen of oyster haemocytes, was shown here to release outer membrane vesicles (OMVs) both in the extracellular milieu and inside haemocytes. Intracellular release of OMVs occurred inside phagosomes of intact haemocytes having phagocytosed few vibrios as well as in damaged haemocytes containing large vacuoles heavily loaded with LGP32. The OMV proteome of LGP32 was shown to be rich in hydrolases (25%) including potential virulence factors such as proteases, lipases, phospholipases, haemolysins and nucleases. One major caseinase/gelatinase named Vsp for vesicular serine protease was found to be specifically secreted through OMVs in which it is enclosed. Vsp was shown to participate in the virulence phenotype of LGP32 in oyster experimental infections. Finally, OMVs were highly protective against antimicrobial peptides, increasing the minimal inhibitory concentration of polymyxin B by 16-fold. Protection was conferred by OMV titration of polymyxin B but did not depend on the activity of Vsp or another OMV-associated protease. Altogether, our results show that OMVs contribute to the pathogenesis of LGP32, being able to deliver virulence factors to host immune cells and conferring protection against antimicrobial peptides.


Assuntos
Ostreidae/microbiologia , Vacúolos/microbiologia , Vibrio/patogenicidade , Fatores de Virulência/metabolismo , Sequência de Aminoácidos , Animais , Antibacterianos/farmacologia , Antígenos de Bactérias/metabolismo , Proteínas da Membrana Bacteriana Externa/metabolismo , Farmacorresistência Bacteriana , Gelatinases/biossíntese , Proteínas Hemolisinas/biossíntese , Metaloendopeptidases/biossíntese , Testes de Sensibilidade Microbiana , Dados de Sequência Molecular , Ostreidae/imunologia , Fagossomos/microbiologia , Polimixina B/farmacologia , Serina Endopeptidases/biossíntese , Serina Proteases/biossíntese , Vibrio/genética
14.
J Biol Chem ; 289(46): 31765-31776, 2014 Nov 14.
Artigo em Inglês | MEDLINE | ID: mdl-25274629

RESUMO

The function of selenium-binding protein 1 (SBP1), present in almost all organisms, has not yet been established. In mammals, SBP1 is known to bind the essential element selenium but the binding site has not been identified. In addition, the SBP family has numerous potential metal-binding sites that may play a role in detoxification pathways in plants. In Arabidopsis thaliana, AtSBP1 over-expression increases tolerance to two toxic compounds for plants, selenium and cadmium, often found as soil pollutants. For a better understanding of AtSBP1 function in detoxification mechanisms, we investigated the chelating properties of the protein toward different ligands with a focus on selenium using biochemical and biophysical techniques. Thermal shift assays together with inductively coupled plasma mass spectrometry revealed that AtSBP1 binds selenium after incubation with selenite (SeO3(2-)) with a ligand to protein molar ratio of 1:1. Isothermal titration calorimetry confirmed the 1:1 stoichiometry and revealed an unexpectedly large value of binding enthalpy suggesting a covalent bond between selenium and AtSBP1. Titration of reduced Cys residues and comparative mass spectrometry on AtSBP1 and the purified selenium-AtSBP1 complex identified Cys(21) and Cys(22) as being responsible for the binding of one selenium. These results were validated by site-directed mutagenesis. Selenium K-edge x-ray absorption near edge spectroscopy performed on the selenium-AtSBP1 complex demonstrated that AtSBP1 reduced SeO3(2-) to form a R-S-Se(II)-S-R-type complex. The capacity of AtSBP1 to bind different metals and selenium is discussed with respect to the potential function of AtSBP1 in detoxification mechanisms and selenium metabolism.


Assuntos
Proteínas de Arabidopsis/química , Arabidopsis/efeitos dos fármacos , Proteínas de Transporte/química , Regulação da Expressão Gênica de Plantas , Proteínas de Ligação a Selênio/química , Selênio/química , Sequência de Aminoácidos , Animais , Sítios de Ligação , Cisteína/química , Humanos , Ligantes , Conformação Molecular , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Ligação Proteica , Proteínas Recombinantes/química , Homologia de Sequência de Aminoácidos , Termodinâmica
15.
J Biol Chem ; 289(36): 24821-31, 2014 Sep 05.
Artigo em Inglês | MEDLINE | ID: mdl-25037219

RESUMO

Although antimicrobial histones have been isolated from multiple metazoan species, their role in host defense has long remained unanswered. We found here that the hemocytes of the oyster Crassostrea gigas release antimicrobial H1-like and H5-like histones in response to tissue damage and infection. These antimicrobial histones were shown to be associated with extracellular DNA networks released by hemocytes, the circulating immune cells of invertebrates, in response to immune challenge. The hemocyte-released DNA was found to surround and entangle vibrios. This defense mechanism is reminiscent of the neutrophil extracellular traps (ETs) recently described in vertebrates. Importantly, oyster ETs were evidenced in vivo in hemocyte-infiltrated interstitial tissues surrounding wounds, whereas they were absent from tissues of unchallenged oysters. Consistently, antimicrobial histones were found to accumulate in oyster tissues following injury or infection with vibrios. Finally, oyster ET formation was highly dependent on the production of reactive oxygen species by hemocytes. This shows that ET formation relies on common cellular and molecular mechanisms from vertebrates to invertebrates. Altogether, our data reveal that ET formation is a defense mechanism triggered by infection and tissue damage, which is shared by relatively distant species suggesting either evolutionary conservation or convergent evolution within Bilateria.


Assuntos
Imunidade Adaptativa/imunologia , Crassostrea/imunologia , Armadilhas Extracelulares/imunologia , Histonas/imunologia , Invertebrados/imunologia , Sequência de Aminoácidos , Animais , Anti-Infecciosos/imunologia , Anti-Infecciosos/metabolismo , Anti-Infecciosos/farmacologia , Bactérias/classificação , Bactérias/efeitos dos fármacos , Crassostrea/metabolismo , Crassostrea/microbiologia , Armadilhas Extracelulares/metabolismo , Hemócitos/imunologia , Hemócitos/metabolismo , Histonas/genética , Histonas/metabolismo , Interações Hospedeiro-Patógeno/imunologia , Invertebrados/metabolismo , Invertebrados/microbiologia , Testes de Sensibilidade Microbiana , Microscopia Confocal , Microscopia de Fluorescência , Dados de Sequência Molecular , Espécies Reativas de Oxigênio/imunologia , Espécies Reativas de Oxigênio/metabolismo , Vibrio/imunologia , Vibrio/fisiologia
16.
PLoS One ; 9(2): e89487, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-24586818

RESUMO

In Toxoplasma gondii, as in other eukaryotes, a subset of the amino-acyl-tRNA synthetases are arranged into an abundant cytoplasmic multi-aminoacyl-tRNA synthetase (MARS) complex. Through a series of genetic pull-down assays, we have identified the enzymes of this complex as: methionyl-, glutaminyl-, glutamyl-, and tyrosyl-tRNA synthetases, and we show that the N-terminal GST-like domain of a partially disordered hybrid scaffold protein, Tg-p43, is sufficient for assembly of the intact complex. Our gel filtration studies revealed significant heterogeneity in the size and composition of isolated MARS complexes. By targeting the tyrosyl-tRNA synthetases subunit, which was found exclusively in the complete 1 MDa complex, we were able to directly visualize MARS particles in the electron microscope. Image analyses of the negative stain data revealed the observed heterogeneity and instability of these complexes to be driven by the intrinsic flexibility of the domain arrangements within the MARS complex. These studies provide unique insights into the assembly of these ubiquitous but poorly understood eukaryotic complexes.


Assuntos
Aminoacil-tRNA Sintetases/metabolismo , Glicoproteínas de Membrana/metabolismo , Proteínas de Protozoários/metabolismo , Proteínas Recombinantes/metabolismo , Toxoplasmose/microbiologia , Sequência de Aminoácidos , Aminoacil-tRNA Sintetases/química , Aminoacil-tRNA Sintetases/genética , Animais , Western Blotting , Cromatografia em Gel , Dicroísmo Circular , Citoplasma/metabolismo , Feminino , Imunofluorescência , Humanos , Imunoprecipitação , Glicoproteínas de Membrana/química , Glicoproteínas de Membrana/genética , Camundongos , Dados de Sequência Molecular , Proteínas de Protozoários/química , Proteínas de Protozoários/genética , Proteínas Recombinantes/genética , Saccharomyces cerevisiae/genética , Homologia de Sequência de Aminoácidos , Taxa de Sobrevida , Toxoplasma/enzimologia , Toxoplasma/patogenicidade , Toxoplasmose/mortalidade , Toxoplasmose/patologia
17.
J Exp Med ; 210(10): 2071-86, 2013 Sep 23.
Artigo em Inglês | MEDLINE | ID: mdl-24043761

RESUMO

Toxoplasma gondii, the causative agent of toxoplasmosis, is an obligate intracellular protozoan parasite that resides inside a parasitophorous vacuole. During infection, Toxoplasma actively remodels the transcriptome of its hosting cells with profound and coupled impact on the host immune response. We report that Toxoplasma secretes GRA24, a novel dense granule protein which traffics from the vacuole to the host cell nucleus. Once released into the host cell, GRA24 has the unique ability to trigger prolonged autophosphorylation and nuclear translocation of the host cell p38α MAP kinase. This noncanonical kinetics of p38α activation correlates with the up-regulation of the transcription factors Egr-1 and c-Fos and the correlated synthesis of key proinflammatory cytokines, including interleukin-12 and the chemokine MCP-1, both known to control early parasite replication in vivo. Remarkably, the GRA24-p38α complex is defined by peculiar structural features and uncovers a new regulatory signaling path distinct from the MAPK signaling cascade and otherwise commonly activated by stress-related stimuli or various intracellular microbes.


Assuntos
Proteínas de Protozoários/imunologia , Toxoplasma/imunologia , Toxoplasmose/imunologia , Toxoplasmose/metabolismo , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo , Motivos de Aminoácidos , Sequência de Aminoácidos , Animais , Linhagem Celular , Núcleo Celular/metabolismo , Quimiocinas/biossíntese , Análise por Conglomerados , Proteína 1 de Resposta de Crescimento Precoce/genética , Proteína 1 de Resposta de Crescimento Precoce/metabolismo , Ativação Enzimática , Feminino , Deleção de Genes , Perfilação da Expressão Gênica , Ordem dos Genes , Humanos , Inflamação/genética , Inflamação/imunologia , Macrófagos/imunologia , Macrófagos/metabolismo , Camundongos , Modelos Moleculares , Dados de Sequência Molecular , Fosforilação , Ligação Proteica , Conformação Proteica , Domínios e Motivos de Interação entre Proteínas , Multimerização Proteica , Transporte Proteico , Proteínas Proto-Oncogênicas c-fos/genética , Proteínas Proto-Oncogênicas c-fos/metabolismo , Proteínas de Protozoários/química , Proteínas de Protozoários/genética , Proteínas de Protozoários/metabolismo , Alinhamento de Sequência , Toxoplasma/genética , Proteínas Quinases p38 Ativadas por Mitógeno/química
18.
J Am Chem Soc ; 135(42): 15841-50, 2013 Oct 23.
Artigo em Inglês | MEDLINE | ID: mdl-24059326

RESUMO

Photobleaching, the irreversible photodestruction of a chromophore, severely limits the use of fluorescent proteins (FPs) in optical microscopy. Yet, the mechanisms that govern photobleaching remain poorly understood. In Reversibly Switchable Fluorescent Proteins (RSFPs), a class of FPs that can be repeatedly photoswitched between nonfluorescent and fluorescent states, photobleaching limits the achievable number of switching cycles, a process known as photofatigue. We investigated the photofatigue mechanisms in the protein IrisFP using combined X-ray crystallography, optical in crystallo spectroscopy, mass spectrometry and modeling approaches. At laser-light intensities typical of conventional wide-field fluorescence microscopy, an oxygen-dependent photobleaching pathway was evidenced. Structural modifications induced by singlet-oxygen production within the chromophore pocket revealed the oxidation of two sulfur-containing residues, Met159 and Cys171, locking the chromophore in a nonfluorescent protonated state. At laser-light intensities typical of localization-based nanoscopy (>0.1 kW/cm(2)), a completely different, oxygen-independent photobleaching pathway was found to take place. The conserved Glu212 underwent decarboxylation concomitantly with an extensive rearrangement of the H-bond network around the chromophore, and an sp(2)-to-sp(3) hybridization change of the carbon atom bridging the chromophore cyclic moieties was observed. This two-regime photobleaching mechanism is likely to be a common feature in RSFPs from Anthozoan species, which typically share high structural and sequence identity with IrisFP. In addition, our results suggest that, when such FPs are used, the illumination conditions employed in localization-based super-resolution microscopy might generate less cytotoxicity than those of standard wide-field microscopy at constant absorbed light-dose. Finally, our data will facilitate the rational design of FPs displaying enhanced photoresistance.


Assuntos
Proteínas Luminescentes/química , Cristalografia por Raios X , Cinética , Proteínas Luminescentes/metabolismo , Modelos Moleculares , Simulação de Dinâmica Molecular , Oxigênio/química , Oxigênio/metabolismo , Fotodegradação , Conformação Proteica
19.
EMBO Mol Med ; 5(8): 1180-95, 2013 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-23828858

RESUMO

Immuno-chemotherapy elicit high response rates in B-cell non-Hodgkin lymphoma but heterogeneity in response duration is observed, with some patients achieving cure and others showing refractory disease or relapse. Using a transcriptome-powered targeted proteomics screen, we discovered a gene regulatory circuit involving the nuclear factor CYCLON which characterizes aggressive disease and resistance to the anti-CD20 monoclonal antibody, Rituximab, in high-risk B-cell lymphoma. CYCLON knockdown was found to inhibit the aggressivity of MYC-overexpressing tumours in mice and to modulate gene expression programs of biological relevance to lymphoma. Furthermore, CYCLON knockdown increased the sensitivity of human lymphoma B cells to Rituximab in vitro and in vivo. Strikingly, this effect could be mimicked by in vitro treatment of lymphoma B cells with a small molecule inhibitor for BET bromodomain proteins (JQ1). In summary, this work has identified CYCLON as a new MYC cooperating factor that autonomously drives aggressive tumour growth and Rituximab resistance in lymphoma. This resistance mechanism is amenable to next-generation epigenetic therapy by BET bromodomain inhibition, thereby providing a new combination therapy rationale for high-risk lymphoma.


Assuntos
Anticorpos Monoclonais Murinos/farmacologia , Antineoplásicos/farmacologia , Redes Reguladoras de Genes , Linfoma de Células B/tratamento farmacológico , Linfoma de Células B/metabolismo , Animais , Antígenos CD20/metabolismo , Azepinas/farmacologia , Diferenciação Celular , Linhagem Celular Tumoral , Núcleo Celular/metabolismo , Proliferação de Células , Epigênese Genética , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Inativação Gênica , Humanos , Linfoma , Camundongos , Camundongos SCID , Transplante de Neoplasias , Estrutura Terciária de Proteína , Proteômica , Rituximab , Triazóis/farmacologia
20.
J Biol Chem ; 288(27): 20085-92, 2013 Jul 05.
Artigo em Inglês | MEDLINE | ID: mdl-23709220

RESUMO

Coenzyme Q (ubiquinone or Q) is a redox-active lipid found in organisms ranging from bacteria to mammals in which it plays a crucial role in energy-generating processes. Q biosynthesis is a complex pathway that involves multiple proteins. In this work, we show that the uncharacterized conserved visC gene is involved in Q biosynthesis in Escherichia coli, and we have renamed it ubiI. Based on genetic and biochemical experiments, we establish that the UbiI protein functions in the C5-hydroxylation reaction. A strain deficient in ubiI has a low level of Q and accumulates a compound derived from the Q biosynthetic pathway, which we purified and characterized. We also demonstrate that UbiI is only implicated in aerobic Q biosynthesis and that an alternative enzyme catalyzes the C5-hydroxylation reaction in the absence of oxygen. We have solved the crystal structure of a truncated form of UbiI. This structure shares many features with the canonical FAD-dependent para-hydroxybenzoate hydroxylase and represents the first structural characterization of a monooxygenase involved in Q biosynthesis. Site-directed mutagenesis confirms that residues of the flavin binding pocket of UbiI are important for activity. With our identification of UbiI, the three monooxygenases necessary for aerobic Q biosynthesis in E. coli are known.


Assuntos
Proteínas de Escherichia coli/metabolismo , Escherichia coli/enzimologia , Flavina-Adenina Dinucleotídeo/metabolismo , Hidrolases/metabolismo , Oxigenases de Função Mista/metabolismo , Ubiquinona/biossíntese , Aerobiose/fisiologia , Sítios de Ligação/fisiologia , Escherichia coli/genética , Proteínas de Escherichia coli/genética , Flavina-Adenina Dinucleotídeo/genética , Hidrolases/genética , Hidroxilação/fisiologia , Oxigenases de Função Mista/genética , Mutagênese Sítio-Dirigida , Ubiquinona/genética
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