RESUMO
Leptospirosis is a widespread zoonosis, potentially severe in humans, caused by spirochetal bacteria, Leptospira interrogans (L. interrogans). Host defense mechanisms involved in leptospirosis are poorly understood. Recognition of lipopolysaccharide (LPS) and lipoproteins by Toll-Like Receptors (TLR)4 and TLR2 is crucial for clearance of leptospires in mice, yet the role of Nucleotide Oligomerization Domain (NOD)-like receptors (NOD)1 and NOD2, recognizing peptidoglycan (PG) fragments has not previously been examined. Here, we show that pathogenic leptospires escape from NOD1 and NOD2 recognition both in vitro and in vivo, in mice. We found that leptospiral PG is resistant to digestion by certain hydrolases and that a conserved outer membrane lipoprotein of unknown function, LipL21, specific for pathogenic leptospires, is tightly bound to the PG. Leptospiral PG prepared from a mutant not expressing LipL21 (lipl21-) was more readily digested than the parental or complemented strains. Muropeptides released from the PG of the lipl21- mutant, or prepared using a procedure to eliminate the LipL21 protein from the PG of the parental strain, were recognized in vitro by the human NOD1 (hNOD1) and NOD2 (hNOD2) receptors, suggesting that LipL21 protects PG from degradation into muropeptides. LipL21 expressed in E. coli also resulted in impaired PG digestion and NOD signaling. We found that murine NOD1 (mNOD1) did not recognize PG of L. interrogans. This result was confirmed by mass spectrometry showing that leptospiral PG was primarily composed of MurTriDAP, the natural agonist of hNOD1, and contained only trace amounts of the tetra muropeptide, the mNOD1 agonist. Finally, in transgenic mice expressing human NOD1 and deficient for the murine NOD1, we showed enhanced clearance of a lipl21- mutant compared to the complemented strain, or to what was observed in NOD1KO mice, suggesting that LipL21 facilitates escape from immune surveillance in humans. These novel mechanisms allowing L. interrogans to escape recognition by the NOD receptors may be important in circumventing innate host responses.
Assuntos
Antígenos de Bactérias/metabolismo , Proteínas da Membrana Bacteriana Externa/metabolismo , Evasão da Resposta Imune , Leptospira interrogans/imunologia , Leptospira interrogans/patogenicidade , Lipoproteínas/metabolismo , Proteína Adaptadora de Sinalização NOD1/imunologia , Proteína Adaptadora de Sinalização NOD2/imunologia , Peptidoglicano/metabolismo , Animais , Antígenos de Bactérias/genética , Antígenos de Bactérias/imunologia , Proteínas da Membrana Bacteriana Externa/genética , Proteínas da Membrana Bacteriana Externa/imunologia , Feminino , Humanos , Evasão da Resposta Imune/genética , Imunidade Inata , Leptospira/imunologia , Leptospira interrogans/genética , Leptospirose/genética , Leptospirose/imunologia , Leptospirose/microbiologia , Lipoproteínas/genética , Lipoproteínas/imunologia , Masculino , Espectrometria de Massas , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Mutação , Proteína Adaptadora de Sinalização NOD1/deficiência , Proteína Adaptadora de Sinalização NOD1/genética , Proteína Adaptadora de Sinalização NOD2/deficiência , Proteína Adaptadora de Sinalização NOD2/genética , Peptidoglicano/química , Peptidoglicano/imunologia , Ligação Proteica , Transdução de Sinais , Especificidade da Espécie , Virulência/genética , Virulência/imunologiaRESUMO
Protein phosphorylation is one of the most studied post-translational modifications that is involved in different cellular events in Leishmania. In this study, we performed a comparative phosphoproteomics analysis of potassium antimonyl tartrate (SbIII)-resistant and -susceptible lines of Leishmania braziliensis using a 2D-DIGE approach followed by MS. In order to investigate the differential phosphoprotein abundance associated with the drug-induced stress response and SbIII-resistance mechanisms, we compared nontreated and SbIII-treated samples of each line. Pair wise comparisons revealed a total of 116 spots that showed a statistically significant difference in phosphoprotein abundance, including 11 and 34 spots specifically correlated with drug treatment and resistance, respectively. We identified 48 different proteins distributed into seven biological process categories. The category "protein folding/chaperones and stress response" is mainly implicated in response to SbIII treatment, while the categories "antioxidant/detoxification," "metabolic process," "RNA/DNA processing," and "protein biosynthesis" are modulated in the case of antimony resistance. Multiple sequence alignments were performed to validate the conservation of phosphorylated residues in nine proteins identified here. Western blot assays were carried out to validate the quantitative phosphoproteome analysis. The results revealed differential expression level of three phosphoproteins in the lines analyzed. This novel study allowed us to profile the L. braziliensis phosphoproteome, identifying several potential candidates for biochemical or signaling networks associated with antimony resistance phenotype in this parasite.
Assuntos
Antimônio/farmacologia , Leishmania braziliensis/efeitos dos fármacos , Leishmania braziliensis/metabolismo , Fosfoproteínas/análise , Eletroforese em Gel Diferencial Bidimensional/métodos , Sequência de Aminoácidos , Simulação por Computador , Resistência a Medicamentos/efeitos dos fármacos , Dados de Sequência Molecular , Fosfoproteínas/metabolismo , Fosforilação , Processamento de Proteína Pós-Traducional/efeitos dos fármacos , Proteínas de Protozoários/análise , Proteínas de Protozoários/metabolismo , Reprodutibilidade dos TestesRESUMO
Protein complexes directing messenger RNA (mRNA) degradation are present in all kingdoms of life. In Escherichia coli, mRNA degradation is performed by an RNA degradosome organized by the major ribonuclease RNase E. In bacteria lacking RNase E, the existence of a functional RNA degradosome is still an open question. Here, we report that in the bacterial pathogen Helicobacter pylori, RNA degradation is directed by a minimal RNA degradosome consisting of Hp-RNase J and the only DExD-box RNA helicase of H. pylori, RhpA. We show that the protein complex promotes faster degradation of double-stranded RNA in vitro in comparison with Hp-RNase J alone. The ATPase activity of RhpA is stimulated in the presence of Hp-RNase J, demonstrating that the catalytic capacity of both partners is enhanced upon interaction. Remarkably, both proteins are associated with translating ribosomes and not with individual 30S and 50S subunits. Moreover, Hp-RNase J is not recruited to ribosomes to perform rRNA maturation. Together, our findings imply that in H. pylori, the mRNA-degrading machinery is associated with the translation apparatus, a situation till now thought to be restricted to eukaryotes and archaea.
Assuntos
Endorribonucleases/metabolismo , Helicobacter pylori/enzimologia , Complexos Multienzimáticos/metabolismo , Polirribonucleotídeo Nucleotidiltransferase/metabolismo , RNA Helicases/metabolismo , RNA Mensageiro/metabolismo , Ribossomos/enzimologia , Adenosina Trifosfatases/metabolismo , Bacillus subtilis/enzimologia , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Endorribonucleases/genética , Endorribonucleases/isolamento & purificação , Helicobacter pylori/genética , Helicobacter pylori/crescimento & desenvolvimento , Proteínas de Membrana Transportadoras/genética , Proteínas de Membrana Transportadoras/metabolismo , Mutação , Biossíntese de Proteínas , RNA Helicases/isolamento & purificação , RNA de Cadeia Dupla/metabolismo , RNA Ribossômico/metabolismoRESUMO
Programmed necrosis induced by DNA alkylating agents, such as MNNG, is a caspase-independent mode of cell death mediated by apoptosis-inducing factor (AIF). After poly(ADP-ribose) polymerase 1, calpain, and Bax activation, AIF moves from the mitochondria to the nucleus where it induces chromatinolysis and cell death. The mechanisms underlying the nuclear action of AIF are, however, largely unknown. We show here that, through its C-terminal proline-rich binding domain (PBD, residues 543-559), AIF associates in the nucleus with histone H2AX. This interaction regulates chromatinolysis and programmed necrosis by generating an active DNA-degrading complex with cyclophilin A (CypA). Deletion or directed mutagenesis in the AIF C-terminal PBD abolishes AIF/H2AX interaction and AIF-mediated chromatinolysis. H2AX genetic ablation or CypA downregulation confers resistance to programmed necrosis. AIF fails to induce chromatinolysis in H2AX or CypA-deficient nuclei. We also establish that H2AX is phosphorylated at Ser139 after MNNG treatment and that this phosphorylation is critical for caspase-independent programmed necrosis. Overall, our data shed new light in the mechanisms regulating programmed necrosis, elucidate a key nuclear partner of AIF, and uncover an AIF apoptogenic motif.
Assuntos
Fator de Indução de Apoptose/metabolismo , Caspases/metabolismo , Cromatina/metabolismo , Histonas/metabolismo , Necrose/metabolismo , Animais , Fator de Indução de Apoptose/química , Calpaína/metabolismo , Linhagem Celular , Ciclofilina A/genética , Ciclofilina A/metabolismo , Dano ao DNA , Regulação para Baixo , Fibroblastos/citologia , Fibroblastos/metabolismo , Deleção de Genes , Histonas/química , Histonas/genética , Metilnitronitrosoguanidina/farmacologia , Camundongos , Modelos Moleculares , Poli(ADP-Ribose) Polimerase-1 , Poli(ADP-Ribose) Polimerases/metabolismo , Proteína X Associada a bcl-2/metabolismoRESUMO
Purified protein quality control is the final and critical check-point of any protein production process. Unfortunately, it is too often overlooked and performed hastily, resulting in irreproducible and misleading observations in downstream applications. In this review, we aim at proposing a simple-to-follow workflow based on an ensemble of widely available physico-chemical technologies, to assess sequentially the essential properties of any protein sample: purity and integrity, homogeneity and activity. Approaches are then suggested to optimize the homogeneity, time-stability and storage conditions of purified protein preparations, as well as methods to rapidly evaluate their reproducibility and lot-to-lot consistency.
Assuntos
Proteínas Recombinantes/química , Proteínas Recombinantes/isolamento & purificação , Controle de Qualidade , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/genéticaRESUMO
Leishmania is exposed to a sudden increase in environmental temperature during the infectious cycle that triggers stage differentiation and adapts the parasite phenotype to intracellular survival in the mammalian host. The absence of classical promoter-dependent mechanisms of gene regulation and constitutive expression of most of the heat-shock proteins (HSPs) in these human pathogens raise important unresolved questions as to regulation of the heat-shock response and stage-specific functions of Leishmania HSPs. Here we used a gel-based quantitative approach to assess the Leishmania donovani phosphoproteome and revealed that 38% of the proteins showed significant stage-specific differences, with a strong focus of amastigote-specific phosphoproteins on chaperone function. We identified STI1/HOP-containing chaperone complexes that interact with ribosomal client proteins in an amastigote-specific manner. Genetic analysis of STI1/HOP phosphorylation sites in conditional sti1(-/-) null mutant parasites revealed two phosphoserine residues essential for parasite viability. Phosphorylation of the major Leishmania chaperones at the pathogenic stage suggests that these proteins may be promising drug targets via inhibition of their respective protein kinases.
Assuntos
Proteínas de Choque Térmico/metabolismo , Leishmania donovani/metabolismo , Fosfoproteínas/metabolismo , Proteoma/metabolismo , Proteínas de Protozoários/metabolismo , Sequência de Aminoácidos , Animais , Humanos , Leishmania donovani/crescimento & desenvolvimento , Dados de Sequência Molecular , Fosfoproteínas/química , Fosforilação , Ligação Proteica , Proteoma/química , Proteínas de Protozoários/química , Alinhamento de SequênciaRESUMO
In eukaryotes, ribosome biogenesis is a highly conserved process that starts in the nucleus and ends in the cytoplasm. In actively growing yeast cells, it is estimated that each nuclear pore complex (NPC) contributes to the export of about 25 pre-ribosomal particles per minute. Such an extremely active process requires several redundant export receptors for the pre-60S particles. Here, we report the identification of a novel pre-60S factor, Ecm1, which partially acts like Arx1 and becomes essential when the NPC function is affected. Ecm1 depletion, combined with the deletion of NPC components led to pre-60S retention in the nucleus. Functional links that we identified between Ecm1, 60S biogenesis, pre-60S export, and the NPC were correlated with physical interactions of Ecm1 with pre-60S particles and nucleoporins. These results support that Ecm1 is an additional factor involved in pre-60S export. While Ecm1 and Arx1 have redundant functions, overproduction of either one could not complement the absence of the other, whereas overproduction of Mex67 was able to partially restore the growth defect resulting from the absence of Ecm1 or Arx1. These data highlight the involvement of many factors acting together to export pre-60S particles.
Assuntos
Precursores de RNA/metabolismo , Subunidades Ribossômicas Maiores de Eucariotos/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Transporte Ativo do Núcleo Celular , Genes Fúngicos , Poro Nuclear/metabolismo , Precursores de RNA/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/crescimento & desenvolvimento , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Técnicas do Sistema de Duplo-Híbrido , beta Carioferinas/genética , beta Carioferinas/metabolismoRESUMO
BACKGROUND: The pathogenesis of Mycobacterium tuberculosis largely depends on the secretion of the 6-kD early secreted antigenic target ESAT-6 (EsxA) and the 10-kD culture filtrate protein CFP-10 (EsxB) via the ESX-1/typeVII secretion system. Although gene products from the core RD1 region have been shown to be deeply implicated in this process, less is known about proteins encoded further upstream in the 5' region of the ESX-1 cluster, such as the ESX-1 secretion-associated proteins (Esps) EspF or EspG(1). METHODS: To elucidate the role of EspF/G(1), whose orthologs in Mycobacterium marinum and Mycobacterium smegmatis are reportedly involved in EsxA/B secretion, we constructed 3 M. tuberculosis knockout strains deleted for espF, espG(1) or the segment corresponding to the combined RD1(bcg)-RD1(mic) region of bacille Calmette-Guérin (BCG) and Mycobacterium microti, which also contains espF and espG(1). RESULTS: Analysis of these strains revealed that, unlike observations with the model organisms M. smegmatis or M. marinum, disruption of espF and espG(1) in M. tuberculosis did not impact the secretion and T cell recognition of EsxA/B but still caused severe attenuation. CONCLUSIONS: The separation of the 2 ESX-1-connected phenotypes (ie, EsxA/B secretion and virulence) indicates that EsxA/B secretion is not the only readout for a functional ESX-1 system and suggests that other processes involving EspF/G(1) also play important roles in ESX-1-mediated pathogenicity.
Assuntos
Proteínas de Bactérias/metabolismo , Proteínas de Transporte/metabolismo , Regulação Bacteriana da Expressão Gênica/fisiologia , Mycobacterium tuberculosis/metabolismo , Mycobacterium tuberculosis/patogenicidade , Animais , Antígenos de Bactérias/genética , Proteínas de Bactérias/genética , Deleção de Genes , Técnicas de Inativação de Genes , Teste de Complementação Genética , Camundongos , Camundongos Endogâmicos C57BL , Família Multigênica , Mycobacterium tuberculosis/genética , Processamento de Proteína Pós-Traducional , VirulênciaRESUMO
Vibrio parahaemolyticus and V. alginolyticus, marine foodborne pathogens, were treated with UVC-radiation (240 J/m(2)) to evaluate alterations in their outer membrane protein profiles. Outer membrane protein patterns of UVC-irradiated bacteria were found altered when analyzed by sodium dodecyl sulphate polyacrylamide gel electrophoresis. Altered proteins were identified by mass spectrometry (MS and MS/MS) and analysis revealed that OmpW, OmpA, Long-chain fatty acid transport protein, Outer membrane receptor protein, Putative uncharacterized protein VP0167, Maltoporin (lamB), Polar flagellin B/D, Agglutination protein Peptidoglycan-associated lipoprotein and MltA-interacting protein MipA were appeared, thereby they can be considered as UVC-stress proteins in some vibrios. In addition, expression of OmpK decreased to non-detectable level. Furthermore, we observed a decrease or an increase in the expression level of other outer membrane proteins.
RESUMO
Interleukin (IL)-7 is a central cytokine that controls homeostasis of the CD4 T lymphocyte pool. Here we show on human primary cells that IL-7 binds to preassembled receptors made up of proprietary chain IL-7Ralpha and the common chain gammac shared with IL-2, -4, -9, -15, and -21 receptors. Upon IL-7 binding, both chains are driven in cholesterol- and sphingomyelin-rich rafts where associated signaling proteins Jak1, Jak3, STAT1, -3, and -5 are found to be phosphorylated. Meanwhile the IL-7.IL-7R complex interacts with the cytoskeleton that halts its diffusion as measured by single molecule fluorescence autocorrelated spectroscopy monitored by microimaging. Comparative immunoprecipitations of IL-7Ralpha signaling complex from non-stimulated and IL-7-stimulated cells confirmed recruitment of proteins such as STATs, but many others were also identified by mass spectrometry from two-dimensional gels. Among recruited proteins, two-thirds are involved in cytoskeleton and raft formation. Thus, early events leading to IL-7 signal transduction involve its receptor compartmentalization into membrane nanodomains and cytoskeleton recruitment.
Assuntos
Linfócitos T CD4-Positivos/imunologia , Compartimento Celular , Interleucina-7/metabolismo , Receptores de Interleucina-7/metabolismo , Transdução de Sinais , Sítios de Ligação , Eletroforese em Gel de Poliacrilamida , Humanos , Imunoprecipitação , Ativação Linfocitária , Espectrometria de Massas , Espectrometria de Fluorescência , UltracentrifugaçãoRESUMO
Ribosome biogenesis is driven by a large number of preribosomal factors that associate with and dissociate from the preribosomal particles along the maturation pathway. We have previously shown that budding yeast Mak11, whose homologues in other eukaryotes were described as modulating a p21-activated protein kinase function, accumulates in Rlp24-associated pre-60S complexes when their maturation is impeded in Saccharomyces cerevisiae. The functional inactivation of WD40 repeat protein Mak11 interfered with the 60S rRNA maturation, led to a cell cycle delay in G(1), and blocked green fluorescent protein-tagged Rpl25 in the nucleoli of yeast cells, indicating an early role of Mak11 in ribosome assembly. Surprisingly, Mak11 inactivation also led to a dramatic destabilization of Rlp24. The suppression of the thermosensitive phenotype of a mak11 mutant by RLP24 overexpression and a direct in vitro interaction between Rlp24 and Mak11 suggest that Mak11 acts as an Rlp24 cofactor during early steps of 60S ribosomal subunit assembly. Moreover, we found that Skb15, the Mak11 homologue in Schizosaccharomyces pombe, also associated with preribosomes and affected 60S biogenesis in fission yeast. It is thus likely that the previously observed phenotypes for MAK11 homologues in other eukaryotes are secondary to the main function of these proteins in ribosome formation.
Assuntos
Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Proteínas Serina-Treonina Quinases/metabolismo , Ribossomos/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Proteínas de Schizosaccharomyces pombe/metabolismo , Schizosaccharomyces/metabolismo , Homologia de Sequência , Sequência de Aminoácidos , Nucléolo Celular/metabolismo , Dados de Sequência Molecular , Complexos Multiproteicos/metabolismo , Proteínas Mutantes/metabolismo , Mutação/genética , Ligação Proteica , Proteínas Serina-Treonina Quinases/química , Precursores de RNA/metabolismo , Proteínas Ribossômicas/metabolismo , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Schizosaccharomyces pombe/química , Quinases Ativadas por p21RESUMO
In the human gastric bacterium Helicobacter pylori, two metalloenzymes, hydrogenase and urease, are essential for in vivo colonization, the latter being a major virulence factor. The UreA and UreB structural subunits of urease and UreG, one of the accessory proteins for Ni(2+) incorporation into apourease, were taken as baits for tandem affinity purification. The method allows the purification of protein complexes under native conditions and physiological expression levels of the bait protein. Furthermore the tandem affinity purification technology was combined with in vivo cross-link to capture transient interactions. The results revealed different populations of urease complexes: (i) urease captured during activation by Ni(2+) ions comprising all the accessory proteins and (ii) urease in association with metabolic proteins involved e.g. in ammonium incorporation and the cytoskeleton. Using UreG as a bait protein, we copurified HypB, the accessory protein for Ni(2+) incorporation into hydrogenase, that is reported to play a role in urease activation. The interactome of HypB partially overlapped with that of urease and revealed interactions with SlyD, which is known to be involved in hydrogenase maturation as well as with proteins implicated in the formation of [Fe-S] clusters present in the small subunit of hydrogenase. In conclusion, this study provides new insight into coupling of ammonium production and assimilation in the gastric pathogen and the intimate link between urease and hydrogenase maturation.
Assuntos
Cromatografia de Afinidade/métodos , Helicobacter pylori/enzimologia , Urease/metabolismo , Proteínas de Bactérias/isolamento & purificação , Proteínas de Bactérias/metabolismo , Cromossomos Bacterianos , Misturas Complexas/metabolismo , Reagentes de Ligações Cruzadas/farmacologia , Estudos de Viabilidade , Glutamato-Amônia Ligase/metabolismo , Helicobacter pylori/efeitos dos fármacos , Hidrogenase/metabolismo , Immunoblotting , Imunoglobulina G/metabolismo , Proteínas Ferro-Enxofre/metabolismo , Metais/metabolismo , Modelos Biológicos , Nitrogênio/metabolismo , Ligação Proteica/efeitos dos fármacos , Proteínas Recombinantes de Fusão/metabolismo , Recombinação Genética/genética , Ureia/metabolismoRESUMO
During the highly conserved process of eukaryotic ribosome formation, RNA follows a maturation path with well-defined, successive intermediates that dynamically associate with many pre-ribosomal proteins. A comprehensive description of the assembly process is still lacking. To obtain data on the timing and order of association of the different pre-ribosomal factors, a strategy consists in the use of pre-ribsomal particles isolated from mutants that block ribosome formation at different steps. Immunoblots, inherently limited to only a few factors, have been applied to evaluate the accumulation or decrease of pre-ribosomal intermediates under mutant conditions. For a global protein-level description of different 60S ribosomal subunit maturation intermediates in yeast, we have adapted a method of in vivo isotopic labelling and mass spectrometry to study pre-60S complexes isolated from strains in which rRNA processing was affected by individual depletion of five factors: Ebp2, Nog1, Nsa2, Nog2 or Pop3. We obtained quantitative data for 45 distinct pre-60S proteins and detected coordinated changes for over 30 pre-60S factors in the analysed mutants. These results led to the characterisation of the composition of early, intermediate and late pre-ribosomal complexes, specific for crucial maturation steps during 60S assembly in eukaryotes.
Assuntos
Proteínas Ribossômicas/metabolismo , Subunidades Ribossômicas Maiores de Eucariotos/química , Subunidades Ribossômicas Maiores de Eucariotos/metabolismo , GTP Fosfo-Hidrolases , Marcação por Isótopo , Espectrometria de Massas , Mutação , Proteínas Ribossômicas/classificação , Proteínas Ribossômicas/genética , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/fisiologiaRESUMO
Mycobacterium ulcerans is the causative agent of Buruli ulcer, a rapidly emerging human disease in which mycolactone, a cytotoxic and immunosuppressive macrocyclic polyketide, is responsible for massive skin destruction. The genome sequencing of M. ulcerans has recently been accomplished (http://genolist.pasteur.fr/BuruList/) enabling the first proteome study of this important human pathogen. Here, we present a comprehensive proteome analysis of different subcellular fractions and culture supernatant of in vitro grown M. ulcerans. By a combination of gel-based and gel-free techniques for protein and peptide separation with subsequent analysis by MS, we identified 1074 different proteins, corresponding to 25% of the protein-coding DNA sequence. Interestingly, new information was obtained about central metabolism and lipid biosynthesis, and as many as 192 conserved hypothetical proteins were found. Comparative analysis of the wild-type strain and an isogenic mycolactone-deficient mutant, by 2-DE and iTRAQ labeling of the cytoplasmic fraction, revealed differences in the expression profiles of proteins involved in lipid metabolism and information pathways, as well as stress responses.
Assuntos
Proteínas de Bactérias/análise , Toxinas Bacterianas/biossíntese , Mycobacterium ulcerans/metabolismo , Proteoma/análise , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Eletroforese em Gel Bidimensional , Macrolídeos , Mycobacterium ulcerans/genética , Proteoma/genética , Proteoma/metabolismo , Proteômica/métodos , Espectrometria de Massas por Ionização por Electrospray , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Espectrometria de Massas em TandemRESUMO
We report global expression profiling of a uvrY-deficient mutant of Photorhabdus luminescens. We found that the regulator moiety of the two-component regulatory system BarA/UvrY regulated more than 500 target genes coding for functions involved in the synthesis of major compartments and metabolic pathways of the cell. This regulation appeared to be in part indirect as UvrY affected the expression of several regulators. Indeed, the flagellum biosynthesis transcription activator FlhC and the flagella regulon were induced in the absence of UvrY, leading to a hyperflagellated phenotype and an increase in motility and biofilm formation. Two major regulatory systems were also altered: the type 2 quorum-sensing inducer AI-2 was activated by UvrY, and the CsrA regulator function appeared to be repressed by the increase of the small-untranslated RNA csrB, the CsrA activity inhibitor TldD and the chaperonin GroESL. Both through and independently of these systems, UvrY regulated oxidative stress resistance; bioluminescence; iron, sugar and peptide transport; proteases; polyketide synthesis enzymes and nucleobases recycling, related to insect degradation and assimilation by bacteria. As a consequence, the uvrY-deficient strain exhibited a decreased killing of insect cells and a reduced growth on insect cells culture, suggesting a UvrY role in the adaptation of P. luminescens inside the insect.
Assuntos
Proteínas de Bactérias/metabolismo , Perfilação da Expressão Gênica , Regulação Bacteriana da Expressão Gênica , Photorhabdus/crescimento & desenvolvimento , Photorhabdus/fisiologia , Spodoptera/microbiologia , Fatores de Transcrição/metabolismo , Adaptação Fisiológica , Animais , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Sequência de Bases , Células Cultivadas , Dados de Sequência Molecular , Mutação , Photorhabdus/genética , Photorhabdus/metabolismo , Percepção de Quorum , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Análise de Sequência de DNA , Transdução de Sinais , Fatores de Transcrição/química , Fatores de Transcrição/genéticaRESUMO
Today, molecular imaging of neurodegenerative diseases is mainly based on small molecule probes. Alternatively, antibodies are versatile tools that may be developed as new imaging agents. Indeed, they can be readily obtained to specifically target any antigen of interest and their scaffold can be functionalized. One of the critical issues involved in translating antibody-based probes to the clinic is the design and synthesis of perfectly-defined conjugates. Camelid single-domain antibody-fragments (VHHs) are very small and stable antibodies that are able to diffuse in tissues and potentially cross the blood brain barrier (BBB). Here, we selected a VHH (R3VQ) specifically targeting one of the main lesions of Alzheimer's disease (AD), namely the amyloid-beta (Aß) deposits. It was used as a scaffold for the design of imaging probes for magnetic resonance imaging (MRI) and labeled with the contrastophore gadolinium using either a random or site-specific approach. In contrast to the random strategy, the site-specific conjugation to a single reduced cysteine in the C-terminal part of the R3VQ generates a well-defined bioconjugate in a high yield process. This new imaging probe is able to cross the BBB and label Aß deposits after intravenous injection. Also, it displays improved r1 and r2 relaxivities, up to 30 times higher than a widely used clinical contrast agent, and it allows MRI detection of amyloid deposits in post mortem brain tissue of a mouse model of AD. The ability to produce chemically-defined VHH conjugates that cross the BBB opens the way for future development of tailored imaging probes targeting intracerebral antigens.
RESUMO
Previous work suggested that the release of the nucleolar Tif6 from nascent 60 S subunits occurs in the cytoplasm and requires the cytoplasmic EF-2-like GTPase, Efl1. To check whether this release involves an rRNA structural rearrangement mediated by Efl1, we analyzed the rRNA conformation of the GTPase center of 80 S ribosomes in three contexts: wild-type, Deltaefl1 and a dominant suppressor R1 of Deltaefl1. This analysis was restricted to domain II and VI of 25 S rRNA. The rRNA analysis of R1 ribosomes allows us to distinguish the effects due to depletion of Efl1 from the resulting nucleolar deficit of Tif6. Efl1 inhibits the EF-2 GTPase activity, suggesting that the two proteins share a similar ribosome-binding site. The 80 S ribosomes from either type failed to show any difference of conformation in the two rRNA domains analyzed. However, the same analysis performed on the pool of free 60 S subunits reveals several rRNA conformational differences between wild-type and Deltaefl1 subunits, whereas that from the suppressor strain is similar to wild-type. This suggests that the nucleolar deficit of Tif6 during assembly of the 60 S preribosomes is responsible for the changes in rRNA conformation observed in Deltaefl1 60 S subunits. We also purified 60 S preribosomes from the three genetic contexts by TAP-tagging Tif6. The protein content of 60 S preribosomes associated with Tif6p in a Deltaefl1 strain are obtained at a lower yield but have, surprisingly, a protein composition that is a priori similar to that of wild-type and the suppressor strain.
Assuntos
Proteínas de Transporte/metabolismo , GTP Fosfo-Hidrolases/metabolismo , Proteínas de Filamentos Intermediários/metabolismo , Fosfoproteínas/metabolismo , RNA Fúngico/metabolismo , RNA Ribossômico/metabolismo , Proteínas Ribossômicas/metabolismo , Ribossomos/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Sítios de Ligação , Proteínas de Transporte/genética , Proteínas de Filamentos Intermediários/genética , Mutação , Conformação de Ácido Nucleico , Fosfoproteínas/genética , RNA Fúngico/genética , RNA Ribossômico/química , RNA Ribossômico/genética , Proteínas Ribossômicas/genética , Ribossomos/genética , Proteínas de Saccharomyces cerevisiae/genética , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por MatrizRESUMO
Detection of intracerebral targets with imaging probes is challenging due to the non-permissive nature of blood-brain barrier (BBB). The present work describes two novel single-domain antibodies (VHHs or nanobodies) that specifically recognize extracellular amyloid deposits and intracellular tau neurofibrillary tangles, the two core lesions of Alzheimer's disease (AD). Following intravenous administration in transgenic mouse models of AD, in vivo real-time two-photon microscopy showed gradual extravasation of the VHHs across the BBB, diffusion in the parenchyma and labeling of amyloid deposits and neurofibrillary tangles. Our results demonstrate that VHHs can be used as specific BBB-permeable probes for both extracellular and intracellular brain targets and suggest new avenues for therapeutic and diagnostic applications in neurology.
Assuntos
Camelídeos Americanos/imunologia , Emaranhados Neurofibrilares/imunologia , Placa Amiloide/imunologia , Anticorpos de Domínio Único/imunologia , Administração Intravenosa , Doença de Alzheimer/metabolismo , Animais , Barreira Hematoencefálica/metabolismo , Encéfalo/metabolismo , Modelos Animais de Doenças , Masculino , Camundongos , Camundongos Transgênicos , Microscopia/métodos , Anticorpos de Domínio Único/metabolismo , Distribuição TecidualRESUMO
TpeL is a toxin produced by Clostridium perfringens which belongs to the large clostridial glucosylating toxin family. It was shown that TpeL modifies Ras using UDP-glucose or UDP-N-acetylglucosamine as cosubstrates (Guttenberg et al., 2012; Nagahama et al., 2011). We confirmed that TpeL preferentially glucosaminates the three isoforms of Ras (cH-Ras, N-Ras, and K-Ras) from UDP-N-acetylglucosamine and to a lower extent Rap1a and R-Ras3, and very weakly Rac1. In contrast to previous report, we observed that Ral was not a substrate of TpeL. In addition, we confirmed by in vitro glucosylation and mass spectrometry that TpeL modifies cH-Ras at Thr35.