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1.
Nat Commun ; 12(1): 4909, 2021 08 13.
Artigo em Inglês | MEDLINE | ID: mdl-34389707

RESUMO

In bacteria, trans-translation is the main rescue system, freeing ribosomes stalled on defective messenger RNAs. This mechanism is driven by small protein B (SmpB) and transfer-messenger RNA (tmRNA), a hybrid RNA known to have both a tRNA-like and an mRNA-like domain. Here we present four cryo-EM structures of the ribosome during trans-translation at resolutions from 3.0 to 3.4 Å. These include the high-resolution structure of the whole pre-accommodated state, as well as structures of the accommodated state, the translocated state, and a translocation intermediate. Together, they shed light on the movements of the tmRNA-SmpB complex in the ribosome, from its delivery by the elongation factor EF-Tu to its passage through the ribosomal A and P sites after the opening of the B1 bridges. Additionally, we describe the interactions between the tmRNA-SmpB complex and the ribosome. These explain why the process does not interfere with canonical translation.


Assuntos
Proteínas de Escherichia coli/genética , Escherichia coli/genética , Biossíntese de Proteínas/genética , RNA Bacteriano/genética , Proteínas de Ligação a RNA/genética , Ribossomos/genética , Sítios de Ligação/genética , Microscopia Crioeletrônica , Escherichia coli/metabolismo , Proteínas de Escherichia coli/metabolismo , Modelos Moleculares , Conformação de Ácido Nucleico , Ligação Proteica , Domínios Proteicos , RNA Bacteriano/química , RNA Bacteriano/metabolismo , RNA Mensageiro/química , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , RNA de Transferência/química , RNA de Transferência/genética , RNA de Transferência/metabolismo , Proteínas de Ligação a RNA/química , Proteínas de Ligação a RNA/metabolismo , Ribossomos/metabolismo , Ribossomos/ultraestrutura
2.
EMBO Rep ; 22(5): e51412, 2021 05 05.
Artigo em Inglês | MEDLINE | ID: mdl-33710763

RESUMO

In the past decades, many studies reported the presence of endoplasmic reticulum (ER)-resident proteins in the cytosol. However, the mechanisms by which these proteins relocate and whether they exert cytosolic functions remain unknown. We find that a subset of ER luminal proteins accumulates in the cytosol of glioblastoma cells isolated from mouse and human tumors. In cultured cells, ER protein reflux to the cytosol occurs upon ER proteostasis perturbation. Using the ER luminal protein anterior gradient 2 (AGR2) as a proof of concept, we tested whether the refluxed proteins gain new functions in the cytosol. We find that refluxed, cytosolic AGR2 binds and inhibits the tumor suppressor p53. These data suggest that ER reflux constitutes an ER surveillance mechanism to relieve the ER from its contents upon stress, providing a selective advantage to tumor cells through gain-of-cytosolic functions-a phenomenon we name ER to Cytosol Signaling (ERCYS).


Assuntos
Degradação Associada com o Retículo Endoplasmático , Retículo Endoplasmático , Animais , Citosol/metabolismo , Retículo Endoplasmático/metabolismo , Estresse do Retículo Endoplasmático , Camundongos , Proteínas/metabolismo
3.
BMC Microbiol ; 20(1): 237, 2020 08 03.
Artigo em Inglês | MEDLINE | ID: mdl-32746783

RESUMO

BACKGROUND: The increase in bacterial resistance phenotype cases is a global health problem. New strategies must be explored by the scientific community in order to create new treatment alternatives. Animal venoms are a good source for antimicrobial peptides (AMPs), which are excellent candidates for new antimicrobial drug development. Cathelicidin-related antimicrobial peptides (CRAMPs) from snake venoms have been studied as a model for the design of new antimicrobial pharmaceuticals against bacterial infections. RESULTS: In this study we present an 11 amino acid-long peptide, named pseudonajide, which is derived from a Pseudonaja textilis venom peptide and has antimicrobial and antibiofilm activity against Staphylococcus epidermidis. Pseudonajide was selected based on the sequence alignments of various snake venom peptides that displayed activity against bacteria. Antibiofilm activity assays with pseudonajide concentrations ranging from 3.12 to 100 µM showed that the lowest concentration to inhibit biofilm formation was 25 µM. Microscopy analysis demonstrated that pseudonajide interacts with the bacterial cell envelope, disrupting the cell walls and membranes, leading to morphological defects in prokaryotes. CONCLUSIONS: Our results suggest that pseudonajide's positives charges interact with negatively charged cell wall components of S. epidermidis, leading to cell damage and inhibiting biofilm formation.

4.
Nucleic Acids Res ; 46(6): 3211-3217, 2018 04 06.
Artigo em Inglês | MEDLINE | ID: mdl-29408956

RESUMO

During translation's elongation cycle, elongation factor G (EF-G) promotes messenger and transfer RNA translocation through the ribosome. Until now, the structures reported for EF-G-ribosome complexes have been obtained by trapping EF-G in the ribosome. These results were based on use of non-hydrolyzable guanosine 5'-triphosphate (GTP) analogs, specific inhibitors or a mutated EF-G form. Here, we present the first cryo-electron microscopy structure of EF-G bound to ribosome in the absence of an inhibitor. The structure reveals a natural conformation of EF-G·GDP in the ribosome, with a previously unseen conformation of its third domain. These data show how EF-G must affect translocation, and suggest the molecular mechanism by which fusidic acid antibiotic prevents the release of EF-G after GTP hydrolysis.


Assuntos
Proteínas de Bactérias/metabolismo , Fator G para Elongação de Peptídeos/metabolismo , Biossíntese de Proteínas , Ribossomos/metabolismo , Proteínas de Bactérias/química , Proteínas de Bactérias/ultraestrutura , Microscopia Crioeletrônica , Guanosina Trifosfato/metabolismo , Hidrólise , Modelos Moleculares , Conformação Molecular , Fator G para Elongação de Peptídeos/química , Fator G para Elongação de Peptídeos/ultraestrutura , Ligação Proteica , Conformação Proteica , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , RNA de Transferência/genética , RNA de Transferência/metabolismo , Ribossomos/química , Ribossomos/ultraestrutura , Thermus thermophilus/metabolismo
5.
Environ Microbiol ; 19(9): 3579-3594, 2017 09.
Artigo em Inglês | MEDLINE | ID: mdl-28695648

RESUMO

Bacterial adhesion is a critical step for colonization of the host. The pioneer colonizer and commensal bacterium of the human gastrointestinal tract, Streptococcus salivarius, has strong adhesive properties but the molecular determinants of this adhesion remain uncharacterized. Serine-rich repeat (SRR) glycoproteins are a family of adhesins that fulfil an important role in adhesion. In general, Gram-positive bacterial genomes have a unique SRR glycoprotein-encoding gene. We demonstrate that S. salivarius expresses three large and glycosylated surface-exposed proteins - SrpA, SrpB and SrpC - that show characteristics of SRR glycoproteins and are secreted through the accessory SecA2/Y2 system. Two glycosyltransferases - GtfE/F - encoded outside of the secA2/Y2 locus, unusually, perform the first step of the sequential glycosylation process, which is crucial for SRR activity. We show that SrpB and SrpC play complementary adhesive roles involved in several steps of the colonization process: auto-aggregation, biofilm formation and adhesion to a variety of host epithelial cells and components. We also show that at least one of the S. salivarius SRR glycoproteins is important for colonization in mice. SrpA, SrpB and SrpC are the main factors underlying the multifaceted adhesion of S. salivarius and, therefore, play a major role in host colonization.


Assuntos
Adesinas Bacterianas/metabolismo , Aderência Bacteriana/fisiologia , Proteínas de Bactérias/metabolismo , Mucosa Intestinal/microbiologia , Glicoproteínas de Membrana/metabolismo , Streptococcus salivarius/patogenicidade , Animais , Aderência Bacteriana/genética , Células Epiteliais/microbiologia , Trato Gastrointestinal/microbiologia , Glucosiltransferases/genética , Glicosilação , Humanos , Masculino , Camundongos , Modelos Animais , Streptococcus salivarius/genética , Streptococcus salivarius/metabolismo
6.
Methods ; 117: 59-66, 2017 03 15.
Artigo em Inglês | MEDLINE | ID: mdl-27729294

RESUMO

Polysomes are macromolecular complexes made up of multiple ribosomes simultaneously translating a single mRNA into polypeptide chains. Together, the cellular mRNAs translated in this way are referred to 'translatome.' Translation determines a cell's overall gene expression profile. Studying translatome leads to a better understanding of the translational machinery and of its complex regulatory pathways. Given its fundamental role in cell homeostasis and division, bacterial translation is an important target for antibiotics. However, there are no detailed protocols for polysome purification from Staphylococcus aureus, the human pathogen responsible for the majority of multi-drug resistance issues. We therefore developed methods for the isolation of active polysomes, ribosomes, and ribosomal subunits, examining the purity and quality of each fraction and monitoring polysomal activity during protein synthesis. These steps are mandatory for the use of purified S. aureus polysomes and ribosomes for structural studies or for genome-scale analysis of most translated mRNAs.


Assuntos
Fracionamento Celular/métodos , Polirribossomos/química , Subunidades Ribossômicas Maiores de Bactérias/química , Subunidades Ribossômicas Menores de Bactérias/química , Staphylococcus aureus/genética , Eletroforese em Gel de Ágar , Microscopia Eletrônica , Polirribossomos/ultraestrutura , Biossíntese de Proteínas , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Subunidades Ribossômicas Maiores de Bactérias/ultraestrutura , Subunidades Ribossômicas Menores de Bactérias/ultraestrutura , Staphylococcus aureus/metabolismo
7.
Mol Biol Cell ; 27(19): 2946-64, 2016 10 01.
Artigo em Inglês | MEDLINE | ID: mdl-27535430

RESUMO

During lactation, mammary epithelial cells secrete huge amounts of milk from their apical side. The current view is that caseins are secreted by exocytosis, whereas milk fat globules are released by budding, enwrapped by the plasma membrane. Owing to the number and large size of milk fat globules, the membrane surface needed for their release might exceed that of the apical plasma membrane. A large-scale proteomics analysis of both cytoplasmic lipid droplets and secreted milk fat globule membranes was used to decipher the cellular origins of the milk fat globule membrane. Surprisingly, differential analysis of protein profiles of these two organelles strongly suggest that, in addition to the plasma membrane, the endoplasmic reticulum and the secretory vesicles contribute to the milk fat globule membrane. Analysis of membrane-associated and raft microdomain proteins reinforces this possibility and also points to a role for lipid rafts in milk product secretion. Our results provide evidence for a significant contribution of the endoplasmic reticulum to the milk fat globule membrane and a role for SNAREs in membrane dynamics during milk secretion. These novel aspects point to a more complex model for milk secretion than currently envisioned.


Assuntos
Glicolipídeos/biossíntese , Glicolipídeos/metabolismo , Glicoproteínas/biossíntese , Glicoproteínas/metabolismo , Animais , Mama/metabolismo , Caseínas/metabolismo , Membrana Celular/metabolismo , Retículo Endoplasmático/metabolismo , Células Epiteliais/metabolismo , Feminino , Humanos , Lactação/metabolismo , Gotículas Lipídicas , Lipídeos , Glândulas Mamárias Animais/metabolismo , Glândulas Mamárias Humanas/metabolismo , Microdomínios da Membrana/metabolismo , Membranas/metabolismo , Camundongos , Leite/metabolismo , Proteômica/métodos , Proteínas SNARE/metabolismo , Vesículas Transportadoras/metabolismo , Vesículas Transportadoras/fisiologia
8.
Sci Rep ; 5: 17146, 2015 Dec 18.
Artigo em Inglês | MEDLINE | ID: mdl-26679898

RESUMO

Although conversion of the cellular form of the prion protein (PrP(C)) into a misfolded isoform is the underlying cause of prion diseases, understanding PrP(C) physiological functions has remained challenging. PrP(C) depletion or overexpression alters the proliferation and differentiation properties of various types of stem and progenitor cells in vitro by unknown mechanisms. Such involvement remains uncertain in vivo in the absence of any drastic phenotype of mice lacking PrP(C). Here, we report PrP(C) enrichment at the base of the primary cilium in stem and progenitor cells from the central nervous system and cardiovascular system of developing mouse embryos. PrP(C) depletion in a neuroepithelial cell line dramatically altered key cilium-dependent processes, such as Sonic hedgehog signalling and α-tubulin post-translational modifications. These processes were also affected over a limited time window in PrP(C)-ablated embryos. Thus, our study reveals PrP(C) as a potential actor in the developmental regulation of microtubule dynamics and ciliary functions.


Assuntos
Cílios/metabolismo , Príons/metabolismo , Tubulina (Proteína)/metabolismo , Animais , Sistema Cardiovascular/metabolismo , Células Cultivadas , Sistema Nervoso Central/metabolismo , Embrião de Mamíferos/metabolismo , Desenvolvimento Embrionário , Proteínas Hedgehog/metabolismo , Camundongos , Microscopia Confocal , Proteínas PrPC/deficiência , Proteínas PrPC/genética , Príons/genética , Processamento de Proteína Pós-Traducional , RNA Mensageiro/metabolismo , Transdução de Sinais , Células-Tronco/citologia , Células-Tronco/metabolismo
9.
Biochim Biophys Acta ; 1854(10 Pt A): 1412-24, 2015 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-26151834

RESUMO

The 90-kDa heat shock protein (Hsp90) is a highly flexible dimer that is able to self-associate in the presence of divalent cations or under heat shock. In a previous work, we focused on the Mg2+-induced oligomerization process of Hsp90, and characterized the oligomers. Combining analytical ultracentrifugation, size-exclusion chromatography coupled to multi-angle laser light scattering and high-mass matrix-assisted laser desorption/ionization time-of-flight mass spectrometry, we studied the interaction of p23 with both Hsp90 dimer and oligomers. Even if p23 predominantly binds the Hsp90 dimer, we demonstrated, for the first time, that p23 is also able to interact with Hsp90 oligomers, shifting the Hsp90 dimer-oligomers equilibrium toward dimer. Our results showed that the Hsp90:p23 binding stoichiometry decreases with the Hsp90 oligomerization degree. Therefore, we propose a model in which p23 would act as a "protein wedge" regarding the Hsp90 dimer closure and the Hsp90 oligomerization process.


Assuntos
Proteínas de Choque Térmico HSP90/química , Oxirredutases Intramoleculares/química , Multimerização Proteica , Animais , Química Encefálica , Carbodi-Imidas/química , Cromatografia em Gel , Reagentes para Ligações Cruzadas/química , Escherichia coli/genética , Escherichia coli/metabolismo , Expressão Gênica , Proteínas de Choque Térmico HSP90/genética , Proteínas de Choque Térmico HSP90/metabolismo , Humanos , Oxirredutases Intramoleculares/genética , Oxirredutases Intramoleculares/metabolismo , Modelos Moleculares , Prostaglandina-E Sintases , Ligação Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Suínos , Ultracentrifugação
10.
BMC Microbiol ; 15: 112, 2015 May 25.
Artigo em Inglês | MEDLINE | ID: mdl-26003173

RESUMO

BACKGROUND: Mechanisms underlying the transition from commensalism to virulence in Enterococcus faecalis are not fully understood. We previously identified the enterococcal leucine-rich protein A (ElrA) as a virulence factor of E. faecalis. The elrA gene is part of an operon that comprises four other ORFs encoding putative surface proteins of unknown function. RESULTS: In this work, we compared the susceptibility to phagocytosis of three E. faecalis strains, including a wild-type (WT), a ΔelrA strain, and a strain overexpressing the whole elr operon in order to understand the role of this operon in E. faecalis virulence. While both WT and ΔelrA strains were efficiently phagocytized by RAW 264.7 mouse macrophages, the elr operon-overexpressing strain showed a decreased capability to be internalized by the phagocytic cells. Consistently, the strain overexpressing elr operon was less adherent to macrophages than the WT strain, suggesting that overexpression of the elr operon could confer E. faecalis with additional anti-adhesion properties. In addition, increased virulence of the elr operon-overexpressing strain was shown in a mouse peritonitis model. CONCLUSIONS: Altogether, our results indicate that overexpression of the elr operon facilitates the E. faecalis escape from host immune defenses.


Assuntos
Proteínas de Bactérias/genética , Enterococcus faecalis/fisiologia , Óperon , Peritonite/microbiologia , Fagocitose , Animais , Aderência Bacteriana , Proteínas de Bactérias/metabolismo , Linhagem Celular , Modelos Animais de Doenças , Enterococcus faecalis/genética , Enterococcus faecalis/patogenicidade , Regulação Bacteriana da Expressão Gênica , Infecções por Bactérias Gram-Positivas/microbiologia , Infecções por Bactérias Gram-Positivas/veterinária , Macrófagos/metabolismo , Camundongos , Virulência
11.
Circulation ; 131(11): 1006-18, 2015 Mar 17.
Artigo em Inglês | MEDLINE | ID: mdl-25593290

RESUMO

BACKGROUND: The vascular remodeling responsible for pulmonary arterial hypertension (PAH) involves predominantly the accumulation of α-smooth muscle actin-expressing mesenchymal-like cells in obstructive pulmonary vascular lesions. Endothelial-to-mesenchymal transition (EndoMT) may be a source of those α-smooth muscle actin-expressing cells. METHODS AND RESULTS: In situ evidence of EndoMT in human PAH was obtained by using confocal microscopy of multiple fluorescent stainings at the arterial level, and by using transmission electron microscopy and correlative light and electron microscopy at the ultrastructural level. Findings were confirmed by in vitro analyses of human PAH and control cultured pulmonary artery endothelial cells. In addition, the mRNA and protein signature of EndoMT was recognized at the arterial and lung level by quantitative real-time polymerase chain reaction and Western blot analyses. We confirmed our human observations in established animal models of pulmonary hypertension (monocrotaline and SuHx). After establishing the first genetically modified rat model linked to BMPR2 mutations (BMPR2(Δ140Ex1/+) rats), we demonstrated that EndoMT is linked to alterations in signaling of BMPR2, a gene that is mutated in 70% of cases of familial PAH and in 10% to 40% of cases of idiopathic PAH. We identified molecular actors of this pathological transition, including twist overexpression and vimentin phosphorylation. We demonstrated that rapamycin partially reversed the protein expression patterns of EndoMT, improved experimental PAH, and decreased the migration of human pulmonary artery endothelial cells, providing the proof of concept that EndoMT is druggable. CONCLUSIONS: EndoMT is linked to alterations in BPMR2 signaling and is involved in the occlusive vas cular remodeling of PAH, findings that may have therapeutic implications.


Assuntos
Transdiferenciação Celular , Células Endoteliais/patologia , Hipertensão Pulmonar/patologia , Mesoderma/patologia , Actinas/biossíntese , Actinas/genética , Animais , Biomarcadores , Receptores de Proteínas Morfogenéticas Ósseas Tipo II/biossíntese , Receptores de Proteínas Morfogenéticas Ósseas Tipo II/genética , Movimento Celular , Células Cultivadas , Modelos Animais de Doenças , Perfilação da Expressão Gênica , Humanos , Hipertensão Pulmonar/induzido quimicamente , Hipertensão Pulmonar/genética , Hipóxia/complicações , Pulmão/irrigação sanguínea , Pulmão/metabolismo , Pulmão/patologia , Monocrotalina/toxicidade , Mutação , RNA Mensageiro/biossíntese , Ratos , Sirolimo/farmacologia , Remodelação Vascular , Vimentina/biossíntese , Vimentina/genética
12.
PLoS One ; 9(12): e115903, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-25549363

RESUMO

Caseins, the main milk proteins, interact with colloidal calcium phosphate to form the casein micelle. The mesostructure of this supramolecular assembly markedly influences its nutritional and technological functionalities. However, its detailed molecular organization and the cellular mechanisms involved in its biogenesis have been only partially established. There is a growing body of evidence to support the concept that α(s1)-casein takes center stage in casein micelle building and transport in the secretory pathway of mammary epithelial cells. Here we have investigated the membrane-associated form of α(s1)-casein in rat mammary epithelial cells. Using metabolic labelling we show that α(s1)-casein becomes associated with membranes at the level of the endoplasmic reticulum, with no subsequent increase at the level of the Golgi apparatus. From morphological and biochemical data, it appears that caseins are in a tight relationship with membranes throughout the secretory pathway. On the other hand, we have observed that the membrane-associated form of α(s1)-casein co-purified with detergent-resistant membranes. It was poorly solubilised by Tween 20, partially insoluble in Lubrol WX, and substantially insoluble in Triton X-100. Finally, we found that cholesterol depletion results in the release of the membrane-associated form of α(s1)-casein. These experiments reveal that the insolubility of α(s1)-casein reflects its partial association with a cholesterol-rich detergent-resistant microdomain. We propose that the membrane-associated form of α(s1)-casein interacts with the lipid microdomain, or lipid raft, that forms within the membranes of the endoplasmic reticulum, for efficient forward transport and sorting in the secretory pathway of mammary epithelial cells.


Assuntos
Caseínas/química , Colesterol/química , Mamíferos/metabolismo , Microdomínios da Membrana/química , Animais , Transporte Biológico , Caseínas/metabolismo , Detergentes/farmacologia , Células Epiteliais/metabolismo , Células Epiteliais/ultraestrutura , Evolução Molecular , Feminino , Lactação , Microdomínios da Membrana/efeitos dos fármacos , Micelas , Ratos Wistar , Especificidade da Espécie
13.
PLoS One ; 9(11): e111556, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-25369064

RESUMO

Once daily milking (ODM) induces a reduction in milk production when compared to twice daily milking (TDM). Unilateral ODM of one udder half and TDM of the other half, enables the study of underlying mechanisms independently of inter-individual variability (same genetic background) and of environmental factors. Our results show that in first-calf heifers three CpG, located 10 kb upstream from the CSN1S1 gene were methylated to 33, 34 and 28%, respectively, after TDM but these levels were higher after ODM, 38, 38 and 33%, respectively. These methylation levels were much lower than those observed in the mammary gland during pregnancy (57, 59 and 50%, respectively) or in the liver (74, 78 and 61%, respectively). The methylation level of a fourth CpG (CpG4), located close by (29% during TDM) was not altered after ODM. CpG4 methylation reached 39.7% and 59.5%, during pregnancy or in the liver, respectively. CpG4 is located within a weak STAT5 binding element, arranged in tandem with a second high affinity STAT5 element. STAT5 binding is only marginally modulated by CpG4 methylation, but it may be altered by the methylation levels of the three other CpG nearby. Our results therefore shed light on mechanisms that help to explain how milk production is almost, but not fully, restored when TDM is resumed (15.1 ± 0.2 kg/day instead of 16.2 ± 0.2 kg/day, p<0.01). The STAT5 elements are 100 bp away from a region transcribed in the antisense orientation, in the mammary gland during lactation, but not during pregnancy or in other reproductive organs (ovary or testes). We now need to clarify whether the transcription of this novel RNA is a consequence of STAT5 interacting with the CSN1S1 distal region, or whether it plays a role in the chromatin structure of this region.


Assuntos
Caseínas/genética , Metilação de DNA , Lactação , Leite/química , Fragmentos de Peptídeos/genética , Animais , Sequência de Bases , Bovinos , Indústria de Laticínios , Feminino , Glândulas Mamárias Animais/ultraestrutura , Dados de Sequência Molecular , Família Multigênica , Transcrição Genética
14.
Biomaterials ; 35(24): 6400-11, 2014 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-24816363

RESUMO

Magnetic hyperthermia mediated by magnetic nanomaterials is one promising antitumoral nanotherapy, particularly for its ability to remotely destroy deep tumors. More and more new nanomaterials are being developed for this purpose, with improved heat-generating properties in solution. However, although the ultimate target of these treatments is the tumor cell, the heating efficiency, and the underlying mechanisms, are rarely studied in the cellular environment. Here we attempt to fill this gap by making systematic measurements of both hyperthermia and magnetism in controlled cell environments, using a wide range of nanomaterials. In particular, we report a systematic fall in the heating efficiency for nanomaterials associated with tumour cells. Real-time measurements showed that this loss of heat-generating power occurred very rapidly, within a matter of minutes. The fall in heating correlated with the magnetic characterization of the samples, demonstrating a complete inhibition of the Brownian relaxation in cellular conditions.


Assuntos
Microambiente Celular , Hipertermia Induzida , Fenômenos Magnéticos , Nanopartículas/química , Nanotecnologia , Anisotropia , Linhagem Celular Tumoral , Sobrevivência Celular , Temperatura Alta , Humanos , Nanopartículas/ultraestrutura , Soluções , Espectrofotometria Atômica
15.
PLoS One ; 9(3): e91766, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-24663075

RESUMO

West Nile Virus (WNV) is a zoonotic mosquito-transmitted flavivirus that can infect and cause disease in mammals including humans. Our study aimed at developing a WNV vectored vaccine based on a fish Novirhabdovirus, the Viral Hemorrhagic Septicemia virus (VHSV). VHSV replicates at temperatures lower than 20°C and is naturally inactivated at higher temperatures. A reverse genetics system has recently been developed in our laboratory for VHSV allowing the addition of genes in the viral genome and the recovery of the respective recombinant viruses (rVHSV). In this study, we have generated rVHSV vectors bearing the complete WNV envelope gene (EWNV) (rVHSV-EWNV) or fragments encoding E subdomains (either domain III alone or domain III fused to domain II) (rVHSV-DIIIWNV and rVHSV-DII-DIIIWNV, respectively) in the VHSV genome between the N and P cistrons. With the objective to enhance the targeting of the EWNV protein or EWNV-derived domains to the surface of VHSV virions, Novirhadovirus G-derived signal peptide and transmembrane domain (SPG and TMG) were fused to EWNV at its amino and carboxy termini, respectively. By Western-blot analysis, electron microscopy observations or inoculation experiments in mice, we demonstrated that both the EWNV and the DIIIWNV could be expressed at the viral surface of rVHSV upon addition of SPG. Every constructs expressing EWNV fused to SPG protected 40 to 50% of BALB/cJ mice against WNV lethal challenge and specifically rVHSV-SPGEWNV induced a neutralizing antibody response that correlated with protection. Surprisingly, rVHSV expressing EWNV-derived domain III or II and III were unable to protect mice against WNV challenge, although these domains were highly incorporated in the virion and expressed at the viral surface. In this study we demonstrated that a heterologous glycoprotein and non membrane-anchored protein, can be efficiently expressed at the surface of rVHSV making this approach attractive to develop new vaccines against various pathogens.


Assuntos
Apresentação do Antígeno , DNA Recombinante/genética , Novirhabdovirus/genética , Novirhabdovirus/imunologia , Proteínas do Envelope Viral/imunologia , Vírus do Nilo Ocidental/fisiologia , Animais , Anticorpos Neutralizantes/biossíntese , Anticorpos Neutralizantes/imunologia , Anticorpos Antivirais/biossíntese , Anticorpos Antivirais/imunologia , Linhagem Celular , Feminino , Vetores Genéticos/genética , Imunização , Camundongos , Camundongos Endogâmicos BALB C , Estrutura Terciária de Proteína , Células Th2/imunologia , Proteínas do Envelope Viral/química , Proteínas do Envelope Viral/genética , Vírus do Nilo Ocidental/imunologia
16.
Exp Mol Pathol ; 96(3): 328-38, 2014 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-24657499

RESUMO

Osteochondrosis (OC) is a developmental bone disorder affecting several mammalian species including the horse. Equine OC is described as a focal disruption of endochondral ossification, leading to osteochondral lesions (osteochondritis dissecans, OCD) that may release free bodies within the joint. OCD lesions trigger joint swelling, stiffness and lameness and affects about 30% of the equine population. OCD is considered as multifactorial but its physiopathology is still poorly understood and genes involved in genetic predisposition are still unknown. Our study compared two healthy and two OC-affected 18-month-old French Trotters diagnosed with OCD lesions at the intermediate ridge of the distal tibia. A comparative shot-gun proteomic analysis of non-wounded cartilage and sub-chondral bone from healthy (healthy samples) and OC-affected foals (predisposed samples) identified 83 and 53 modulated proteins, respectively. These proteins are involved in various biological pathways including matrix structure and maintenance, protein biosynthesis, folding and transport, mitochondrial activity, energy and calcium metabolism. Transmission electron microscopy revealed typical features of mitochondrial swelling and ER-stress, such as large, empty mitochondria, and hyper-dilated rough endoplasmic reticulum, in the deep zone of both OC lesions and predisposed cartilage. Abnormal fibril organization surrounding chondrocytes and abnormal features at the ossification front were also observed. Combining these findings with quantitative trait loci and whole genome sequencing results identified about 140 functional candidate genes carrying putative damaging mutations in 30 QTL regions. In summary, our study suggests that OCD lesions may result from defective hypertrophic terminal differentiation associated with mitochondrial dysfunction and ER-stress, leading to impaired cartilage and bone biomechanical properties, making them prone to fractures. In addition, 11 modulated proteins and several candidate mutations located in QTL regions were identified, bringing new insight into the molecular physiopathology and genetic basis of OCD.


Assuntos
Estresse do Retículo Endoplasmático , Mitocôndrias/patologia , Osteocondrite Dissecante/fisiopatologia , Osteocondrite Dissecante/veterinária , Animais , Cartilagem/fisiopatologia , Cartilagem/ultraestrutura , Condrócitos/patologia , Condrócitos/ultraestrutura , Cavalos , Articulações/fisiopatologia , Articulações/ultraestrutura , Microscopia Eletrônica de Transmissão , Mitocôndrias/ultraestrutura , Osteocondrite Dissecante/genética , Osteogênese , Proteômica , Locos de Características Quantitativas , Tíbia/fisiopatologia , Tíbia/ultraestrutura
17.
J Mammary Gland Biol Neoplasia ; 19(1): 119-30, 2014 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-24264376

RESUMO

During lactation, polarized mammary epithelial secretory cells (MESCs) secrete huge quantities of the nutrient molecules that make up milk, i.e. proteins, fat globules and soluble components such as lactose and minerals. Some of these nutrients are only produced by the MESCs themselves, while others are to a great extent transferred from the blood. MESCs can thus be seen as a crossroads for both the uptake and the secretion with cross-talks between intracellular compartments that enable spatial and temporal coordination of the secretion of the milk constituents. Although the physiology of lactation is well understood, the molecular mechanisms underlying the secretion of milk components remain incompletely characterized. Major milk proteins, namely caseins, are secreted by exocytosis, while the milk fat globules are released by budding, being enwrapped by the apical plasma membrane. Prolactin, which stimulates the transcription of casein genes, also induces the production of arachidonic acid, leading to accelerated casein transport and/or secretion. Because of their ability to form complexes that bridge two membranes and promote their fusion, SNARE (Soluble N-ethylmaleimide-Sensitive Factor Attachment Protein Receptor) proteins are involved in almost all intracellular trafficking steps and exocytosis. As SNAREs can bind arachidonic acid, they could be the effectors of the secretagogue effect of prolactin in MESCs. Indeed, some SNAREs have been observed between secretory vesicles and lipid droplets suggesting that these proteins could not only orchestrate the intracellular trafficking of milk components but also act as key regulators for both the coupling and coordination of milk product secretion in response to hormones.


Assuntos
Lactação/metabolismo , Glândulas Mamárias Animais/fisiologia , Glândulas Mamárias Humanas/fisiologia , Leite/metabolismo , Proteínas SNARE/metabolismo , Animais , Feminino , Humanos , Glândulas Mamárias Animais/metabolismo , Glândulas Mamárias Humanas/metabolismo
18.
Mol Cell Proteomics ; 12(12): 3935-47, 2013 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-24002364

RESUMO

Surface proteins of Gram-positive bacteria play crucial roles in bacterial adhesion to host tissues. Regarding commensal or probiotic bacteria, adhesion to intestinal mucosa may promote their persistence in the gastro-intestinal tract and their beneficial effects to the host. In this study, seven Lactococcus lactis strains exhibiting variable surface physico-chemical properties were compared for their adhesion to Caco-2 intestinal epithelial cells. In this test, only one vegetal isolate TIL448 expressed a high-adhesion phenotype. A nonadhesive derivative was obtained by plasmid curing from TIL448, indicating that the adhesion determinants were plasmid-encoded. Surface-exposed proteins in TIL448 were analyzed by a proteomic approach consisting in shaving of the bacterial surface with trypsin and analysis of the released peptides by LC-MS/MS. As the TIL448 complete genome sequence was not available, the tryptic peptides were identified by a mass matching approach against a database including all Lactococcus protein sequences and the sequences deduced from partial DNA sequences of the TIL448 plasmids. Two surface proteins, encoded by plasmids in TIL448, were identified as candidate adhesins, the first one displaying pilin characteristics and the second one containing two mucus-binding domains. Inactivation of the pilin gene abolished adhesion to Caco-2 cells whereas inactivation of the mucus-binding protein gene had no effect on adhesion. The pilin gene is located inside a cluster of four genes encoding two other pilin-like proteins and one class-C sortase. Synthesis of pili was confirmed by immunoblotting detection of high molecular weight forms of pilins associated to the cell wall as well as by electron and atomic force microscopy observations. As a conclusion, surface proteome analysis allowed us to detect pilins at the surface of L. lactis TIL448. Moreover we showed that pili appendages are formed and involved in adhesion to Caco-2 intestinal epithelial cells.


Assuntos
Proteínas de Bactérias/genética , Proteínas de Fímbrias/genética , Fímbrias Bacterianas/genética , Regulação Bacteriana da Expressão Gênica , Lactococcus lactis/genética , Proteoma/genética , Adesinas Bacterianas/genética , Adesinas Bacterianas/metabolismo , Sequência de Aminoácidos , Aminoaciltransferases/genética , Aminoaciltransferases/metabolismo , Aderência Bacteriana , Proteínas de Bactérias/metabolismo , Células CACO-2 , Cromatografia Líquida , Cisteína Endopeptidases/genética , Cisteína Endopeptidases/metabolismo , Proteínas de Fímbrias/metabolismo , Fímbrias Bacterianas/metabolismo , Fímbrias Bacterianas/ultraestrutura , Humanos , Intestinos/citologia , Intestinos/microbiologia , Lactococcus lactis/metabolismo , Lactococcus lactis/ultraestrutura , Microscopia Eletrônica , Anotação de Sequência Molecular , Dados de Sequência Molecular , Família Multigênica , Fragmentos de Peptídeos/análise , Plasmídeos , Probióticos/química , Proteólise , Proteoma/metabolismo , Espectrometria de Massas em Tandem , Tripsina/química
19.
Micron ; 52-53: 16-23, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-23962686

RESUMO

Most studies using microwave irradiation (MWI) for the preparation of tissue samples have reported an improvement in structural integrity. However, there have been few studies on the effect of microwave (MW) on antigen preservation during sample preparation prior to immunolocalization. This report documents our experience of specimen preparation using an automatic microwave apparatus to obtain antigen preservation and retrieval. We tested the effects of MW processing vs. conventional procedures on the morphology and antigenicity of two different tissues: the brain and mammary gland, whose chemical composition and anatomical organization are quite different. We chose to locate the transcription factor PPARß/δ using immunocytochemistry on brain tissue sections from hamsters. Antigen retrieval protocols involving MWI were used to restore immunoreactivity. We also studied the efficiency of the ultrastructural immunolocalization of both PPARγ and caveolin-1 following MWI vs. conventional treatment, on mammary gland tissue from mice at 10 days of lactation. Our findings showed that the treatment of tissue samples with MWI, in the context of a process lasting just a few hours from fixation to immunolocalization, enabled similar, or even better, results than conventional protocols. The quantification of immunolabeling for cav-1 indicated an increase in density of up to three-fold in tissues processed in the microwave oven. Furthermore, MW treatment permitted the localization of PPARß/δ in glutaraldehyde-fixed specimens, which was impossible in the absence of MWI. This study thus showed that techniques involving the use of microwaves could largely improve both ultrastructure and immunodetection.


Assuntos
Antígenos/análise , Microscopia Eletrônica de Transmissão/métodos , Micro-Ondas , Preservação Biológica/métodos , Manejo de Espécimes/métodos , Animais , Automação Laboratorial/métodos , Encéfalo/ultraestrutura , Química Encefálica , Caveolina 1/análise , Cricetinae , Glândulas Mamárias Animais/química , Glândulas Mamárias Animais/ultraestrutura , Camundongos , PPAR delta/análise , PPAR gama/análise , PPAR beta/análise
20.
Nanoscale ; 5(23): 11374-84, 2013 Dec 07.
Artigo em Inglês | MEDLINE | ID: mdl-23827988

RESUMO

There is a great deal of interest in the development of nanoplatforms gathering versatility and multifunctionality. The strategy reported herein meets these requirements and further integrates a cell-friendly shell in a bio-inspired approach. By taking advantage of a cell mechanism of biomolecule transport using vesicles, we engineered a hybrid biogenic nanoplatform able to encapsulate a set of nanoparticles regardless of their chemistry or shape. As a proof of versatility, different types of hybrid nanovesicles were produced: magnetic, magnetic-metallic and magnetic-fluorescent vesicles, either a single component or multiple components, combining the advantageous properties of each integrant nanoparticle. These nanoparticle-loaded vesicles can be manipulated, monitored by MRI and/or fluorescence imaging methods, while acting as efficient nano-heaters. The resulting assets for targeting, imaging and therapy converge for the outline of a new generation of nanosystems merging versatility and multifunctionality into a bio-camouflaged and bio-inspired approach.


Assuntos
Nanoestruturas/química , Lipossomas Unilamelares/química , Materiais Biocompatíveis/química , Materiais Biocompatíveis/metabolismo , Meios de Contraste/química , Meios de Contraste/metabolismo , Compostos Férricos/química , Corantes Fluorescentes/química , Ouro/química , Células Endoteliais da Veia Umbilical Humana , Humanos , Imageamento por Ressonância Magnética , Magnetismo , Nanopartículas Metálicas/química , Microscopia Eletrônica de Transmissão , Tamanho da Partícula , Temperatura , Lipossomas Unilamelares/metabolismo
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