RESUMO
BACKGROUND: Giant viruses have brought new insights into different aspects of virus-cell interactions. The resulting cytopathic effects from these interactions are one of the main aspects of infection assessment in a laboratory routine, mainly reflecting on the morphological features of an infected cell. OBJECTIVES: In this work, we follow the entire kinetics of the cytopathic effect in cells infected by viruses of the Mimiviridae family, spatiotemporally quantifying typical features such as cell roundness, loss of motility, decrease in cell area and cell lysis. METHODS: Infections by Acanthamoeba polyphaga mimivirus (APMV), Tupanvirus (TPV) and M4 were carried out at multiplicity of infection (MOI) 1 and MOI 10 in Acanthamoeba castellanii. Monitoring of infections was carried out using time lapse microscopy for up to 72 hours. The images were analyzed using ImageJ software. FINDINGS: The data obtained indicate that APMV is the slowest virus in inducing the cytopathic effects of rounding, decrease in cell area, mobility and cell lysis. However, it is the only virus whose MOI increase accelerates the lysis process of infected cells. In turn, TPV and M4 rapidly induce morphological and behavioral changes. MAIN CONCLUSIONS: Our results indicate that mimiviruses induce different temporal responses within the host cell and that it is possible to use these kinetic data to facilitate the understanding of infection by these viruses.
Assuntos
Acanthamoeba castellanii , Efeito Citopatogênico Viral , Mimiviridae , Mimiviridae/fisiologia , Cinética , Acanthamoeba castellanii/virologiaRESUMO
BACKGROUND Giant viruses have brought new insights into different aspects of virus-cell interactions. The resulting cytopathic effects from these interactions are one of the main aspects of infection assessment in a laboratory routine, mainly reflecting on the morphological features of an infected cell. OBJECTIVES In this work, we follow the entire kinetics of the cytopathic effect in cells infected by viruses of the Mimiviridae family, spatiotemporally quantifying typical features such as cell roundness, loss of motility, decrease in cell area and cell lysis. METHODS Infections by Acanthamoeba polyphaga mimivirus (APMV), Tupanvirus (TPV) and M4 were carried out at multiplicity of infection (MOI) 1 and MOI 10 in Acanthamoeba castellanii. Monitoring of infections was carried out using time lapse microscopy for up to 72 hours. The images were analyzed using ImageJ software. FINDINGS The data obtained indicate that APMV is the slowest virus in inducing the cytopathic effects of rounding, decrease in cell area, mobility and cell lysis. However, it is the only virus whose MOI increase accelerates the lysis process of infected cells. In turn, TPV and M4 rapidly induce morphological and behavioral changes. MAIN CONCLUSIONS Our results indicate that mimiviruses induce different temporal responses within the host cell and that it is possible to use these kinetic data to facilitate the understanding of infection by these viruses.
RESUMO
The Salmonella enterica bacteriophage P22 is one of the most promising models for the development of virus-like particle (VLP) nanocages. It possesses an icosahedral T = 7 capsid, assembled by the combination of two structural proteins: the coat protein (gp5) and the scaffold protein (gp8). The P22 capsid has the remarkable capability of undergoing structural transition into three morphologies with differing diameters and wall-pore sizes. These varied morphologies can be explored for the design of nanoplatforms, such as for the development of cargo internalization strategies. The capsid proteic nature allows for the extensive modification of its structure, enabling the addition of non-native structures to alter the VLP properties or confer them to diverse ends. Various molecules were added to the P22 VLP through genetic, chemical, and other means to both the capsid and the scaffold protein, permitting the encapsulation or the presentation of cargo. This allows the particle to be exploited for numerous purposes-for example, as a nanocarrier, nanoreactor, and vaccine model, among other applications. Therefore, the present review intends to give an overview of the literature on this amazing particle.
Assuntos
Bacteriófago P22 , Viroides , Capsídeo , Proteínas do Capsídeo/genética , Núcleo Celular , NanotecnologiaRESUMO
Almost two decades after the isolation of the first amoebal giant viruses, indubitably the discovery of these entities has deeply affected the current scientific knowledge on the virosphere. Much has been uncovered since then: viruses can now acknowledge complex genomes and huge particle sizes, integrating remarkable evolutionary relationships that date as early as the emergence of life on the planet. This year, a decade has passed since the first studies on giant viruses in the Brazilian territory, and since then biomes of rare beauty and biodiversity (Amazon, Atlantic forest, Pantanal wetlands, Cerrado savannas) have been explored in the search for giant viruses. From those unique biomes, novel viral entities were found, revealing never before seen genomes and virion structures. To celebrate this, here we bring together the context, inspirations, and the major contributions of independent Brazilian research groups to summarize the accumulated knowledge about the diversity and the exceptionality of some of the giant viruses found in Brazil.
Assuntos
Amoeba/virologia , Vírus Gigantes/genética , Vírus Gigantes/isolamento & purificação , Virologia/história , Biodiversidade , Brasil , Ecossistema , Genoma Viral , Vírus Gigantes/classificação , Vírus Gigantes/ultraestrutura , História do Século XXI , FilogeniaRESUMO
Although giant viruses have existed for millennia and possibly exerted great evolutionary influence in their environment. Their presence has only been noticed by virologists recently with the discovery of Acanthamoeba polyphaga mimivirus in 2003. Its virion with a diameter of 500 nm and its genome larger than 1 Mpb shattered preconceived standards of what a virus is and triggered world-wide prospection studies. Thanks to these investigations many giant virus families were discovered, each with its own morphological peculiarities and genomes ranging from 0.4 to 2.5 Mpb that possibly encode more than 400 viral proteins. This review aims to present the morphological diversity, the different aspects observed in host-virus interactions during replication, as well as the techniques utilized during their investigation.
Assuntos
Amébidos/virologia , Vírus Gigantes/fisiologia , Vírus Gigantes/ultraestrutura , Interações entre Hospedeiro e Microrganismos , Acanthamoeba castellanii/virologia , Genoma Viral , Vírus Gigantes/classificação , Vírus Gigantes/genética , Proteínas Virais/genética , Proteínas Virais/metabolismo , Compartimentos de Replicação Viral/fisiologia , Vírion/fisiologia , Vírion/ultraestrutura , Replicação ViralRESUMO
In Brazil, the use of transgenic plants expressing the insect-toxic Bacillus thuringiensis endotoxin has been successfully used as pest control management since 2013 in transgenic soybean lineages against pest caterpillars such as Helicoverpa armigera. These toxins, endogenously expressed by the plants or sprayed over the crops, are ingested by the insect and bind to receptors in the midgut of these animals, resulting in disruption of digestion and lower insect survival rates. Here, we identified and characterized a membrane-associated alkaline phosphatase (ALP) in the midgut of Anticarsia gemmatalis, the main soybean defoliator pest in Brazil, and data suggested that it binds to Cry1Ac toxin in vitro. Our data showed a peak of ALP activity in homogenate samples of the midgut dissected from the 4th and 5th instars larvae. The brush border membrane vesicles obtained from the midgut of these larvae were used to purify a 60 kDa ALP, as detected by in-gel activity and in vitro biochemical characterization using pharmacological inhibitors and mass spectrometry. When Cry1Ac toxin was supplied to the diet, it was efficient in decreasing larval weight gain and survival. Indeed, in vitro incubation of Cry1Ac toxin with the purified ALP resulted in a 43% decrease in ALP specific activity and enzyme-linked immunosorbent assay showed that ALP interacts with Cry1Ac toxin in vitro, thus suggesting that ALP could function as a Cry toxin ligand. This is a first report characterizing an ALP in A. gemmatalis.
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Fosfatase Alcalina/metabolismo , Proteínas de Bactérias/metabolismo , Endotoxinas/metabolismo , Proteínas Hemolisinas/metabolismo , Larva/enzimologia , Mariposas/enzimologia , Fosfatase Alcalina/antagonistas & inibidores , Fosfatase Alcalina/isolamento & purificação , Animais , Toxinas de Bacillus thuringiensis , Proteínas de Bactérias/toxicidade , Endotoxinas/toxicidade , Trato Gastrointestinal/enzimologia , Trato Gastrointestinal/ultraestrutura , Proteínas Hemolisinas/toxicidade , Microvilosidades/enzimologiaRESUMO
Free-living amoebae (FLAs) are major reservoirs for a variety of bacteria, viruses, and fungi. The most studied mycophagic FLA, Acanthamoeba castellanii (Ac), is a potential environmental host for endemic fungal pathogens such as Cryptococcus spp., Histoplasma capsulatum, Blastomyces dermatitides, and Sporothrix schenckii. However, the mechanisms involved in this interaction are poorly understood. The aim of this work was to characterize the molecular instances that enable Ac to interact with and ingest fungal pathogens, a process that could lead to selection and maintenance of possible virulence factors. The interaction of Ac with a variety of fungal pathogens was analysed in a multifactorial evaluation that included the role of multiplicity of infection over time. Fungal binding to Ac surface by living image consisted of a quick process, and fungal initial extrusion (vomocytosis) was detected from 15 to 80 min depending on the organism. When these fungi were cocultured with the amoeba, only Candida albicans and Cryptococcus neoformans were able to grow, whereas Paracoccidioides brasiliensis and Sporothrix brasiliensis displayed unchanged viability. Yeasts of H. capsulatum and Saccharomyces cerevisiae were rapidly killed by Ac; however, some cells remained viable after 48 hr. To evaluate changes in fungal virulence upon cocultivation with Ac, recovered yeasts were used to infect Galleria mellonella, and in all instances, they killed the larvae faster than control yeasts. Surface biotinylated extracts of Ac exhibited intense fungal binding by FACS and fluorescence microscopy. Binding was also intense to mannose, and mass spectrometry identified Ac proteins with affinity to fungal surfaces including two putative transmembrane mannose-binding proteins (MBP, L8WXW7 and MBP1, Q6J288). Consistent with interactions with such mannose-binding proteins, Ac-fungi interactions were inhibited by mannose. These MBPs may be involved in fungal recognition by amoeba and promotes interactions that allow the emergence and maintenance of fungal virulence for animals.
Assuntos
Acanthamoeba castellanii/metabolismo , Fungos/patogenicidade , Lectina de Ligação a Manose/metabolismo , Acanthamoeba castellanii/química , Acanthamoeba castellanii/microbiologia , Acanthamoeba castellanii/ultraestrutura , Animais , Candida albicans/patogenicidade , Candida albicans/ultraestrutura , Concanavalina A/metabolismo , Cryptococcus neoformans/patogenicidade , Cryptococcus neoformans/ultraestrutura , Histoplasma/patogenicidade , Histoplasma/ultraestrutura , Interações Hospedeiro-Patógeno , Larva/microbiologia , Lepidópteros/microbiologia , Manose/química , Manose/metabolismo , Lectina de Ligação a Manose/química , Espectrometria de Massas , Microscopia Eletrônica de Varredura , Paracoccidioides/patogenicidade , Paracoccidioides/ultraestrutura , Saccharomyces cerevisiae/patogenicidade , Saccharomyces cerevisiae/ultraestrutura , Fatores de Tempo , Imagem com Lapso de Tempo , Virulência , Fatores de Virulência/metabolismoRESUMO
BACKGROUND: Salsolinol (SALSO), a product from the reaction of dopamine (DA) with acetaldehyde, is found increased in dopaminergic neurons of Parkinson's disease (PD) patients. The administration of SALSO in rats causes myenteric neurodegeneration followed by the formation of deposits of the protein α-synuclein (aS), whose aggregation is intimately associated to PD. METHODS: NMR, isothermal titration calorimetry and MS were used to evaluate the interaction of SALSO with aS. The toxicity of SALSO and in vitro-produced aS-SALSO species was evaluated on mesencephalic primary neurons from mice. RESULTS: SALSO, under oxidative conditions, stabilizes the monomeric state besides a minor population of oligomers of aS, resulting in a strong inhibition of the fibrillation process. SALSO does not promote any chemical modification of the protein. Instead, the interaction of SALSO with aS seems to occur via hydrophobic effect, likely mediated by the NAC (non-amyloid component) domain of the protein. aS-SALSO species were found to be innocuous on primary neurons, while SALSO alone induces apoptosis via caspase-3 activation. Importantly, exogenous aS monomer was capable of protecting neurons against SALSO toxicity irrespective whether the protein was co-administered with SALSO or added until 2â¯h after SALSO, as evidenced by DAPI and cleaved-caspase 3 assays. Similar protective action of aS was found by pre-incubating neurons with aS before the administration of SALSO. CONCLUSIONS: Interaction of SALSO with aS leads to the formation of fibril-incompetent and innocuous adducts. SALSO toxicity is attenuated by aS monomer. SIGNIFICANCE: aS could exhibit a protective role against the neurotoxic effects of SALSO in dopaminergic neuron.
Assuntos
Neurônios Dopaminérgicos/efeitos dos fármacos , Isoquinolinas/toxicidade , Sinapses/metabolismo , alfa-Sinucleína/fisiologia , Animais , Apoptose/efeitos dos fármacos , Calorimetria , Caspase 3/metabolismo , Células Cultivadas , Cromatografia em Gel , Cromatografia Líquida de Alta Pressão , Eletroforese em Gel de Poliacrilamida , Ativação Enzimática , Humanos , Espectrometria de Massas , Camundongos , Oxirredução , Ratos , Espectrometria de Fluorescência , alfa-Sinucleína/metabolismoRESUMO
The dopamine metabolite 3,4-dihydroxyphenylacetaldehyde (DOPAL) is believed to play a central role in Parkinson's disease neurodegeneration by stabilizing potentially toxic oligomers of the presynaptic protein α-Synuclein (aSyn). Besides the formation of covalent DOPAL-Lys adducts, DOPAL promotes the oxidation of Met residues of aSyn, which is also a common oxidative post-translational modification found in the protein in vivo. Herein we set out to address the role of Met residues on the oligomerization and neurotoxic properties of DOPAL-modified aSyn. Our data indicate that DOPAL promotes the formation of two distinct types of aSyn oligomers: large and small (dimer and trimers) oligomers, which seem to be generated by independent mechanisms and cannot be interconverted by using denaturing agents. Interestingly, H2O2-treated aSyn monomer, which exhibits all-four Met residues oxidized to Met-sulfoxide, exhibited a reduced ability to form large oligomers upon treatment with DOPAL, with no effect on the population of small oligomers. In this context, triple Met-Val mutant M5V/M116V/M127V exhibited an increased population of large aSyn-DOPAL oligomers in comparison with the wild-type protein. Interestingly, the stabilization of large rather than small oligomers seems to be associated with an enhanced toxicity of DOPAL-aSyn adducts. Collectively, these findings indicate that Met residues may play an important role in modulating both the oligomerization and the neurotoxic properties of DOPAL-derived aSyn species.
Assuntos
Ácido 3,4-Di-Hidroxifenilacético/farmacologia , Metionina/química , Neurônios/efeitos dos fármacos , Multimerização Proteica/efeitos dos fármacos , alfa-Sinucleína/toxicidade , Ácido 3,4-Di-Hidroxifenilacético/análogos & derivados , Animais , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Humanos , Peróxido de Hidrogênio/farmacologia , Metionina/genética , Camundongos , Mutação , Neurônios/citologia , Oxidantes/farmacologia , Oxirredução/efeitos dos fármacos , Processamento de Proteína Pós-Traducional/efeitos dos fármacos , alfa-Sinucleína/química , alfa-Sinucleína/genéticaRESUMO
Acanthamoeba castellanii (Ac) are ubiquitously distributed in nature, and by contaminating medical devices such as heart valves and contact lenses, they cause a broad range of clinical presentations to humans. Although several molecules have been described to play a role in Ac pathogenesis, including parasite host-tissue invasion and escaping of host-defense, little information is available on their mechanisms of secretion. Herein, we describe the molecular components secreted by Ac, under different protein availability conditions to simulate host niches. Ac extracellular vesicles (EVs) were morphologically and biochemically characterized. Dynamic light scattering analysis of Ac EVs identified polydisperse populations, which correlated to electron microscopy measurements. High-performance thin liquid chromatography of Ac EVs identified phospholipids, steryl-esters, sterol and free-fatty acid, the last two also characterized by GC-MS. Secretome composition (EVs and EVs-free supernatants) was also determined and proteins biological functions classified. In peptone-yeast-glucose (PYG) medium, a total of 179 proteins were identified (21 common proteins, 89 exclusive of EVs and 69 in EVs-free supernatant). In glucose alone, 205 proteins were identified (134 in EVs, 14 common and 57 proteins in EVs-free supernatant). From those, stress response, oxidative and protein and amino acid metabolism proteins prevailed. Qualitative differences were observed on carbohydrate metabolism enzymes from Krebs cycle and pentose phosphate shunt. Serine proteases and metalloproteinases predominated. Analysis of the cytotoxicity of Ac EVs (upon uptake) and EVs-free supernatant to epithelial and glioblastoma cells revealed a dose-dependent effect. Therefore, the Ac secretome differs depending on nutrient conditions, and is also likely to vary during infection.
Assuntos
Acanthamoeba castellanii/metabolismo , Amebíase/parasitologia , Vesículas Extracelulares/metabolismo , Proteoma/metabolismo , Proteínas de Protozoários/metabolismo , Acanthamoeba castellanii/genética , Animais , Linhagem Celular , Vesículas Extracelulares/genética , Homeostase , Humanos , Transporte Proteico , Proteoma/genética , Proteômica , Proteínas de Protozoários/genética , Via SecretóriaRESUMO
BACKGROUND: Since the discovery of giant viruses infecting amoebae in 2003, many dogmas of virology have been revised and the search for these viruses has been intensified. Over the last few years, several new groups of these viruses have been discovered in various types of samples and environments.In this work, we describe the isolation of 68 giant viruses of amoeba obtained from environmental samples from Brazil and Antarctica. METHODS: Isolated viruses were identified by hemacolor staining, PCR assays and electron microscopy (scanning and/or transmission). RESULTS: A total of 64 viruses belonging to the Mimiviridae family were isolated (26 from lineage A, 13 from lineage B, 2 from lineage C and 23 from unidentified lineages) from different types of samples, including marine water from Antarctica, thus being the first mimiviruses isolated in this extreme environment to date. Furthermore, a marseillevirus was isolated from sewage samples along with two pandoraviruses and a cedratvirus (the third to be isolated in the world so far). CONCLUSIONS: Considering the different type of samples, we found a higher number of viral groups in sewage samples. Our results reinforce the importance of prospective studies in different environmental samples, therefore improving our comprehension about the circulation anddiversity of these viruses in nature.
Assuntos
Microbiologia Ambiental , Vírus Gigantes/genética , Vírus Gigantes/isolamento & purificação , Amoeba , Animais , Regiões Antárticas , Brasil , DNA Viral , Genoma Viral , Geografia , Vírus Gigantes/classificação , Vírus Gigantes/ultraestrutura , Humanos , Filogenia , Análise de Sequência de DNARESUMO
The inclusion of Mimiviridae members in the putative monophyletic nucleocytoplasmic large DNA virus (NCLDV) group is based on genomic and phylogenomic patterns. This shows that, along with other viral families, they share a set of genes known as core or "hallmark genes," including the gene for the major capsid protein (MCP). Although previous studies have suggested that the maturation of mimivirus MCP transcripts is dependent on splicing, there is little information about the processing of this transcript in other mimivirus isolates. Here we report the characterization of a new mimivirus isolate, called Kroon virus (KV) mimivirus. Analysis of the structure, synteny, and phylogenetic relationships of the MCP genes in many mimivirus isolates revealed a remarkable variation at position and types of intronic and exonic regions, even for mimiviruses belonging to the same lineage. In addition, sequencing of KV and Acanthamoeba polyphaga mimivirus (APMV) MCP transcripts has shown that inside the family, even related giant viruses may present different ways to process the MCP mRNA. These results contribute to the understanding of the genetic organization and evolution of the MCP gene in mimiviruses.IMPORTANCE Mimivirus isolates have been obtained by prospecting studies since 2003. Based on genomic and phylogenomic studies of conserved genes, these viruses have been clustered together with members of six other viral families. Although the major capsid protein (MCP) gene is an important member of the so-called "hallmark genes," there is little information about the processing and structure of this gene in many mimivirus isolates. In this work, we have analyzed the structure, synteny, and phylogenetic relationships of the MCP genes in many mimivirus isolates; these genes showed remarkable variation at position and types of intronic and exonic regions, even for mimiviruses belonging to the same lineage. These results contribute to the understanding of the genetic organization and evolution of the MCP gene in mimiviruses.
Assuntos
Proteínas do Capsídeo/genética , Evolução Molecular , Regulação Viral da Expressão Gênica , Mimiviridae/genética , Splicing de RNA , Transcrição Gênica , Genoma Viral , Mimiviridae/classificação , Mimiviridae/isolamento & purificação , Mimiviridae/ultraestrutura , Filogenia , RNA Viral , Replicação Viral , Microbiologia da ÁguaRESUMO
Prior to the discovery of the mimivirus in 2003, viruses were thought to be physically small and genetically simple. Mimivirus, with its ~750-nm particle size and its ~1.2-Mbp genome, shattered these notions and changed what it meant to be a virus. Since this discovery, the isolation and characterization of giant viruses has exploded. One of the more recently discovered giant viruses, Samba virus, is a Mimivirus that was isolated from the Rio Negro in the Brazilian Amazon. Initial characterization of Samba has revealed some structural information, although the preparation techniques used are prone to the generation of structural artifacts. To generate more native-like structural information for Samba, we analyzed the virus through cryo-electron microscopy, cryo-electron tomography, scanning electron microscopy, and fluorescence microscopy. These microscopy techniques demonstrated that Samba particles have a capsid diameter of ~527 nm and a fiber length of ~155 nm, making Samba the largest Mimivirus yet characterized. We also compared Samba to a fiberless mimivirus variant. Samba particles, unlike those of mimivirus, do not appear to be rigid, and quasi-icosahedral, although the two viruses share many common features, including a multi-layered capsid and an asymmetric nucleocapsid, which may be common amongst the Mimiviruses.
Assuntos
Mimiviridae/ultraestrutura , Brasil , Capsídeo/ultraestrutura , Microscopia , Mimiviridae/isolamento & purificação , Rios/virologiaRESUMO
RhlR is a 241-residue quorum sensing receptor that controls the expression of a myriad of virulence genes in Pseudomonas aeruginosa. Here, the DNA sequence encoding the carboxi-terminal DNA-binding domain of RhlR was cloned into the pET-RP1B plasmid and expressed as an N-terminal fusion protein to the expression/purification Thio6His6 tag. The fusion construct expressed insolubly in Escherichia coli BL21 (DE3) cells. The recombinant protein was extracted from the bacterial inclusion bodies and refolded in the presence of the charged amino acids l-arginine and l-glutamate. The refolded protein was purified by a combination of Ni(+2)-affinity and size exclusion chromatography, allowing the production of 2 mg of highly purified protein (>95% purity) per 5 mg of wet cells derived from 1 L culture. (1)H 1D NMR analysis revealed that the recombinant protein is folded. Moreover, a fluorescence anisotropy DNA-binding assay showed that the refolded protein is functional, as it recognizes the rhlAB promoter. This is the first time that a domain of the quorum sensing regulator RhlR was produced in sufficient amounts for structural studies, enabling the investigation of the molecular basis for RhlR specific interaction with DNA promoters.
Assuntos
Proteínas de Bactérias/química , Proteínas de Ligação a DNA/química , Percepção de Quorum/genética , Proteínas de Bactérias/genética , Proteínas de Bactérias/isolamento & purificação , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/isolamento & purificação , Escherichia coli/genética , Regulação Bacteriana da Expressão Gênica , Regiões Promotoras Genéticas , Dobramento de Proteína , Pseudomonas aeruginosa/genéticaRESUMO
UNLABELLED: Acanthamoeba polyphaga mimivirus (APMV) is a giant virus from the Mimiviridae family. It has many unusual features, such as a pseudoicosahedral capsid that presents a starfish shape in one of its vertices, through which the â¼ 1.2-Mb double-stranded DNA is released. It also has a dense glycoprotein fibril layer covering the capsid that has not yet been functionally characterized. Here, we verified that although these structures are not essential for viral replication, they are truly necessary for viral adhesion to amoebae, its natural host. In the absence of fibrils, APMV had a significantly lower level of attachment to the Acanthamoeba castellanii surface. This adhesion is mediated by glycans, specifically, mannose and N-acetylglucosamine (a monomer of chitin and peptidoglycan), both of which are largely distributed in nature as structural components of several organisms. Indeed, APMV was able to attach to different organisms, such as Gram-positive bacteria, fungi, and arthropods, but not to Gram-negative bacteria. This prompted us to predict that (i) arthropods, mainly insects, might act as mimivirus dispersers and (ii) by attaching to other microorganisms, APMV could be ingested by amoebae, leading to the successful production of viral progeny. To date, this mechanism has never been described in the virosphere. IMPORTANCE: APMV is a giant virus that is both genetically and structurally complex. Its size is similar to that of small bacteria, and it replicates inside amoebae. The viral capsid is covered by a dense glycoprotein fibril layer, but its function has remained unknown, until now. We found that the fibrils are not essential for mimivirus replication but that they are truly necessary for viral adhesion to the cell surface. This interaction is mediated by glycans, mainly N-acetylglucosamine. We also verified that APMV is able to attach to bacteria, fungi, and arthropods. This indicates that insects might act as mimivirus dispersers and that adhesion to other microorganisms could facilitate viral ingestion by amoebae, a mechanism never before described in the virosphere.
Assuntos
Acanthamoeba/virologia , Glicoproteínas/metabolismo , Mimiviridae/fisiologia , Proteínas Virais/metabolismo , Ligação Viral , Acanthamoeba/fisiologia , Acanthamoeba/ultraestrutura , Acetilglucosamina/metabolismo , Análise de Variância , Manose/metabolismo , Microscopia Eletrônica de Transmissão , Especificidade da Espécie , Replicação Viral/fisiologiaRESUMO
Lipases are triacyl glycerol acyl hydrolases, which catalyze hydrolysis of esters, esterification and transesterification reactions, among others. Some of these enzymes have a large hydrophobic pocket covered by an alpha-helical mobile surface loop (the lid). Protein-protein interactions can occur through adsorption of two open lids of individual lipases. We investigated the conformation and oligomeric state of Thermomyces lanuginosus lipase (TLL) in solution by spectroscopic and mass spectrometry techniques. Information about oligomerization of this important industrial enzyme is only available for TLL crystals; therefore, we have done a throughout investigation of the conformation of this lipase in solution. SDS-PAGE and mass spectrometry analysis of size-exclusion chromatography eluted fractions indicated the presence of both monomeric and dimeric populations of TLL. The stability of the enzyme upon thermal and guanidine hydrochloride treatment was examined by circular dichroism and fluorescence emission spectroscopy. Small angle x-ray scattering and ion mobility mass spectrometry analysis revealed that TLL is found as a mixture of monomers and dimers at the assayed concentrations. Although previous x-ray diffraction data showed TLL as a dimer in the crystal (PDB: 1DT3), to our knowledge our report is the first evidencing that TLL co-exists as stable dimeric and monomeric forms in solution.
Assuntos
Ascomicetos/enzimologia , Lipase/química , Ascomicetos/química , Dicroísmo Circular , Espectrometria de Massas , Modelos Moleculares , Multimerização Proteica , Espalhamento a Baixo Ângulo , Espectrometria de Fluorescência , Difração de Raios XRESUMO
Fibrillization of the protein α-synuclein (α-syn) is a hallmark of Parkinson's disease and other α-synucleinopathies. The well-established idea that α-syn is a natively disordered monomer prone to forming fibrils was recently challenged by data showing that the protein mostly exists in vitro and in vivo as helically folded tetramers that are resistant to fibrillization. These apparently conflicting findings may be reconciled by the idea that α-syn exists as a disordered monomer in equilibrium with variable amounts of dynamic oligomeric species. In this context, varying the approaches used for protein purification, such as the method used to lyse cells or the inclusion of denaturing agents, could dramatically perturb this equilibrium and hence alter the relative abundance of the disordered monomer. In the present study, we investigated how the current methods for α-syn purification affect the structure and oligomeric state of the protein, and we discuss the main pitfalls associated with the production of recombinant α-syn in Escherichia coli. We demonstrate that α-syn was expressed in E. coli as a disordered monomer independent of both the cell lysis method and the use of heating/acidification for protein purification. In addition, we provide convincing evidence that the disordered monomer exists in equilibrium with a dynamic dimer, which is not an artefact of the cross-linking protocol as previously suggested. Unlike the helically folded tetramer, α-syn dimer is prone to fibrillate and thus it may be an interesting target for anti-fibrillogenic molecules.
Assuntos
Escherichia coli/metabolismo , alfa-Sinucleína/química , alfa-Sinucleína/metabolismo , Dicroísmo Circular , Escherichia coli/genética , Espectroscopia de Ressonância Magnética , Multimerização ProteicaRESUMO
The head of the P22 bacteriophage is interrupted by a unique dodecameric portal vertex that serves as a conduit for the entrance and exit of the DNA. Here, the in vitro unfolding/refolding processes of the portal protein of P22 were investigated at different temperatures (1, 25, and 37 degrees C) through the use of urea and high hydrostatic pressure (HHP) combined with spectroscopic techniques. We have characterized an intermediate species, IU, which forms at 25 degrees C during unfolding or refolding of the portal protein in 2-4 M urea. IU readily forms amorphous aggregates, rendering the folding process irreversible. On the other hand, at 1 degrees C, a two-state process is observed (DeltaGf = -2.2 kcal/mol). When subjected to HHP at 25 or 37 degrees C, the portal monomer undergoes partial denaturation, also forming an intermediate species, which we call IP. IP also tends to aggregate but, differently from IU, aggregates into a ring-like structure as seen by size-exclusion chromatography and electron microscopy. Again, at 1 degrees C the unfolding induced by HHP proved to be reversible, with DeltaGf = -2.4 kcal/mol and DeltaV = 72 mL/mol. Interestingly, at 25 degrees C, the binding of the hydrophobic probe bis-ANS to the native portal protein destabilizes it and completely blocks its aggregation under HHP. These data are relevant to the process by which the portal protein assembles into dodecamers in vivo, since species such as IP must prevail over IU in order to guarantee the proper ring formation.