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Int J Mol Sci ; 22(12)2021 Jun 21.
Artigo em Inglês | MEDLINE | ID: mdl-34205737


3-bromopuryvate (3-BP) is a compound with unique antitumor activity. It has a selective action against tumor cells that exhibit the Warburg effect. It has been proven that the action of 3-BP is pleiotropic: it acts on proteins, glycolytic enzymes, reduces the amount of ATP, induces the formation of ROS (reactive oxygen species), and induces nuclear DNA damage. Mitochondria are important organelles for the proper functioning of the cell. The production of cellular energy (ATP), the proper functioning of the respiratory chain, or participation in the production of amino acids are one of the many functions of mitochondria. Here, for the first time, we show on the yeast model that 3-BP acts in the eukaryotic cell also by influence on mitochondria and that agents inhibiting mitochondrial function can potentially be used in cancer therapy with 3-BP. We show that cells with functional mitochondria are more resistant to 3-BP than rho0 cells. Using an MTT assay (a colorimetric assay for assessing cell metabolic activity), we demonstrated that 3-BP decreased mitochondrial activity in yeast in a dose-dependent manner. 3-BP induces mitochondrial-dependent ROS generation which results in ∆sod2, ∆por1, or ∆gpx1 mutant sensitivity to 3-BP. Probably due to ROS mtDNA lesions rise during 3-BP treatment. Our findings may have a significant impact on the therapy with 3-BP.

Antineoplásicos/farmacologia , DNA Mitocondrial/efeitos dos fármacos , Mitocôndrias/efeitos dos fármacos , Piruvatos/farmacologia , Avaliação Pré-Clínica de Medicamentos , Mitocôndrias/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Saccharomyces cerevisiae
Molecules ; 25(19)2020 Sep 24.
Artigo em Inglês | MEDLINE | ID: mdl-32987777


For the first time, we are introducing TTPBgp12 and TFPgp17 as new members of the tail tubular proteins B (TTPB) and tail fiber proteins (TFP) family, respectively. These proteins originate from Yersinia enterocolitica phage φYeO3-12. It was originally thought that these were structural proteins. However, our results show that they also inhibit bacterial growth and biofilm formation. According to the bioinformatic analysis, TTPBgp12 is functionally and structurally similar to the TTP of Enterobacteria phage T7 and adopts a ß-structure. TFPgp17 contains an intramolecular chaperone domain at its C-terminal end. The N-terminus of TFPgp17 is similar to other representatives of the TFP family. Interestingly, the predicted 3D structure of TFPgp17 is similar to other bacterial S-layer proteins. Based on the thermal unfolding experiment, TTPBgp12 seems to be a two-domain protein that aggregates in the presence of sugars such as maltose and N-acetylglucosamine (GlcNAc). These sugars cause two unfolding events to transition into one global event. TFPgp17 is a one-domain protein. Maltose and GlcNAc decrease the aggregation temperature of TFPgp17, while the presence of N-acetylgalactosamine (GalNAc) increases the temperature of its aggregation. The thermal unfolding analysis of the concentration gradient of TTPBgp12 and TFPgp17 indicates that with decreasing concentrations, both proteins increase in stability. However, a decrease in the protein concentration also causes an increase in its aggregation, for both TTPBgp12 and TFPgp17.

Caudovirales , Proteínas Estruturais Virais , Yersinia enterocolitica/virologia , Caudovirales/química , Caudovirales/genética , Caudovirales/metabolismo , Domínios Proteicos , Proteínas Estruturais Virais/química , Proteínas Estruturais Virais/genética , Proteínas Estruturais Virais/metabolismo
Front Microbiol ; 11: 1356, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32636826


We report here the complete genome sequence and characterization of Yersinia bacteriophage vB_YenP_ϕ80-18. ϕ80-18 was isolated in 1991 using a Y. enterocolitica serotype O:8 strain 8081 as a host from a sewage sample in Turku, Finland, and based on its morphological and genomic features is classified as a podovirus. The genome is 42 kb in size and has 325 bp direct terminal repeats characteristic for podoviruses. The genome contains 57 predicted genes, all encoded in the forward strand, of which 29 showed no similarity to any known genes. Phage particle proteome analysis identified altogether 24 phage particle-associated proteins (PPAPs) including those identified as structural proteins such as major capsid, scaffolding and tail component proteins. In addition, also the DNA helicase, DNA ligase, DNA polymerase, 5'-exonuclease, and the lytic glycosylase proteins were identified as PPAPs, suggesting that they might be injected together with the phage genome into the host cell to facilitate the take-over of the host metabolism. The phage-encoded RNA-polymerase and DNA-primase were not among the PPAPs. Promoter search predicted the presence of four phage and eleven host RNA polymerase -specific promoters in the genome, suggesting that early transcription of the phage is host RNA-polymerase dependent and that the phage RNA polymerase takes over later. The phage tolerates pH values between 2 and 12, and is stable at 50°C but is inactivated at 60°C. It grows slowly with a 50 min latent period and has apparently a low burst size. Electron microscopy revealed that the phage has a head diameter of about 60 nm, and a short tail of 20 nm. Whole-genome phylogenetic analysis confirmed that ϕ80-18 belongs to the Autographivirinae subfamily of the Podoviridae family, that it is 93.2% identical to Yersinia phage fHe-Yen3-01. Host range analysis showed that ϕ80-18 can infect in addition to Y. enterocolitica serotype O:8 strains also strains of serotypes O:4, O:4,32, O:20 and O:21, the latter ones representing similar to Y. enterocolitica serotype O:8, the American pathogenic biotype 1B strains. In conclusion, the phage ϕ80-18 is a promising candidate for the biocontrol of the American biotype 1B Y. enterocolitica.

Sci Rep ; 10(1): 4196, 2020 03 06.
Artigo em Inglês | MEDLINE | ID: mdl-32144374


Tail Tubular Protein A (TTPA) was long thought to be strictly a structural protein of environmental bacteriophages. However, our recent work has suggested that some TTPAs have additional functional features and thus are dual-function proteins. This study introduces a new TTPA family member, TTPAgp11, which belongs to Yersinia phage phiYeO3-12. We cloned the gene, expressed it and then purified the phage protein. The protein, including its hydrolytic activity, was characterized. Our enzymatic activity tests showed that TTPAgp11 displayed hydrolytic activity towards Red-starch, suggesting that this enzyme could be classified as part as the α - 1, 4-glucosidase family. Protein folding and aggregation tests indicated that TTPAgp11 is a single-domain protein whose aggregation can be induced by maltose or N-acetylglucosamine. The spatial structure of TTPAgp11 seemed to resemble that of the first reported dual-function TTPA, TTPAgp31, which was isolated from Klebsiella pneumoniae phage 32.

Proteínas Virais/química , Proteínas Virais/metabolismo , Sequência de Aminoácidos , Bacteriófagos/genética , Genoma Viral/genética , Hidrólise , Dados de Sequência Molecular , Proteínas Virais/genética
New Phytol ; 228(3): 973-988, 2020 11.
Artigo em Inglês | MEDLINE | ID: mdl-33410187


Plasma membrane (PM) intrinsic proteins (PIPs) are aquaporins facilitating the diffusion of water and small solutes. The functional importance of the PM organisation of PIPs in the interaction with other cellular structures is not completely understood. We performed a pull-down assay using maize (Zea mays) suspension cells expressing YFP-ZmPIP2;5 and validated the protein interactions by yeast split-ubiquitin and bimolecular fluorescence complementation assays. We expressed interacting proteins tagged with fluorescent proteins in Nicotiana benthamiana leaves and performed water transport assays in oocytes. Finally, a phylogenetic analysis was conducted. The PM-located ZmPIP2;5 physically interacts with the endoplasmic reticulum (ER) resident ZmVAP27-1. This interaction requires the ZmVAP27-1 cytoplasmic major sperm domain. ZmPIP2;5 and ZmVAP27-1 localise in close vicinity in ER-PM contact sites (EPCSs) and endocytic structures upon exposure to salt stress conditions. This interaction enhances PM water permeability in oocytes. Similarly, the Arabidopsis ZmVAP27-1 paralogue, AtVAP27-1, interacts with the AtPIP2;7 aquaporin. Together, these data indicate that the PIP2-VAP27 interaction in EPCSs is evolutionarily conserved, and suggest that VAP27 might stabilise the aquaporins and guide their endocytosis in response to salt stress.

Aquaporinas , Retículo Endoplasmático , Aquaporinas/genética , Membrana Celular , Oócitos , Filogenia