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In the present study, we compared the genetic variability of fragments from the internal transcribed spacer region (ITS) and the small subunit ribosomal DNA (SSUrDNA) as nuclear markers, in contrast with the ribosomal protein large two (rpl2) loci, placed in the mitochondrion-related organelles (MROs) within and among human fecal samples with Blastocystis. Samples were analyzed using polymerase chain reaction (PCR)-sequencing, phylogenies, and genetics of population structure analyses were performed. In total, 96 sequences were analyzed, i.e., 33 of SSUrDNA, 35 of rpl2, and 28 of ITS. Only three subtypes (STs) were identified, i.e., ST1 (11.4%), ST2 (28.6%), and ST3 (60%); in all cases, kappa indexes were 1, meaning a perfect agreement among ST assignations. The topologies of phylogenetic inferences were similar among them, clustering to each ST in its specific cluster; discrepancies between phylogeny and assignment of STs were not observed. The STRUCTURE v2.3.4 software assigned three subpopulations corresponding to the STs 1-3, respectively. The population indices were consistent with those previously reported by other groups. Our results suggest the potential use of the ITS and rpl2 genes as molecular markers for Blastocystis subtyping as an alternative approach for the study of the genetic diversity observed within and between human isolates of this microorganism.
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Rhipicephalus (Boophilus) microplus, also known as the cattle tick, causes severe parasitism and transmits different pathogens to vertebrate hosts, leading to massive economic losses. In the present study, we performed a functional characterization of a ribosomal protein from R. microplus to investigate its importance in blood feeding, egg production and viability. Ribosomal protein S18 (RPS18) is part of the 40S subunit, associated with 18S rRNA, and has been previously pointed to have a secondary role in different organisms. Rhipicephalus microplus RPS18 (RmRPS18) gene expression levels were modulated in female salivary glands during blood feeding. Moreover, mRNA levels in this tissue were 10 times higher than those in the midgut of fully engorged female ticks. Additionally, recombinant RmRPS18 was recognized by IgG antibodies from sera of cattle naturally or experimentally infested with ticks. RNAi-mediated knockdown of the RmRPS18 gene was performed in fully engorged females, leading to a significant (29 %) decrease in egg production. Additionally, egg hatching was completely impaired, suggesting that no viable eggs were produced by the RmRPS18-silenced group. Furthermore, antimicrobial assays revealed inhibitory activities against gram-negative Escherichia coli and gram-positive Staphylococcus aureus bacteria, affecting bacterial growth. Data presented here show the important role of RmRPS18 in tick physiology and suggest that RmRPS18 can be a potential target for the development of novel strategies for tick control.
Assuntos
Proteínas de Artrópodes , Rhipicephalus , Proteínas Ribossômicas , Animais , Rhipicephalus/genética , Rhipicephalus/fisiologia , Proteínas Ribossômicas/genética , Feminino , Bovinos , Proteínas de Artrópodes/genética , Proteínas de Artrópodes/metabolismo , Doenças dos Bovinos/parasitologia , Glândulas SalivaresRESUMO
BACKGROUND: Hepatocellular carcinoma (HCC) is one of the most common types of malignant tumors, with a slow onset, rapid progression, and frequent recurrence. Previous research has implicated mitochondrial ribosomal genes in the development, metastasis, and prognosis of various cancers. However, further research is necessary to establish a link between mitochondrial ribosomal protein (MRP) family expression and HCC diagnosis, prognosis, ferroptosis-related gene (FRG) expression, m6A modification-related gene expression, tumor immunity, and drug sensitivity. METHODS: Bioinformatics resources were used to analyze data from patients with HCC retrieved from the TCGA, ICGC, and GTEx databases (GEPIA, UALCAN, Xiantao tool, cBioPortal, STRING, Cytoscape, TISIDB, and GSCALite). RESULTS: Among the 82 MRP family members, 14 MRP genes (MRPS21, MRPS23, MRPL9, DAP3, MRPL13, MRPL17, MRPL24, MRPL55, MRPL16, MRPL14, MRPS17, MRPL47, MRPL21, and MRPL15) were significantly upregulated differentially expressed genes (DEGs) in HCC tumor samples in comparison to normal samples. Receiver-operating characteristic curve analysis indicated that all 14 DEGs show good diagnostic performance. Furthermore, TCGA analysis revealed that the mRNA expression of 39 MRPs was associated with overall survival (OS) in HCC. HCC was divided into two molecular subtypes (C1 and C2) with distinct prognoses using clustering analysis. The clusters showed different FRG expression and m6A methylation profiles and immune features, and prognostic models showed that the model integrating 5 MRP genes (MRPS15, MRPL3, MRPL9, MRPL36, and MRPL37) and 2 FRGs (SLC1A5 and SLC5A11) attained a greater clinical net benefit than three other prognostic models. Finally, analysis of the CTRP and GDSC databases revealed several potential drugs that could target prognostic MRP genes. CONCLUSION: We identified 14 MRP genes as HCC diagnostic markers. We investigated FRG and m6A modification-related gene expression profiles and immune features in patients with HCC, and developed and validated a model incorporating MRP and FRG expression that accurately and reliably predicts HCC prognosis and may predict disease progression and treatment response.
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Carcinoma Hepatocelular , Neoplasias Hepáticas , Humanos , Carcinoma Hepatocelular/genética , Neoplasias Hepáticas/genética , Prognóstico , Ribossomos , Proteínas Ribossômicas/genética , Biomarcadores Tumorais/genética , Antígenos de Histocompatibilidade Menor , Sistema ASC de Transporte de Aminoácidos , Proteínas de Transporte de Sódio-GlucoseRESUMO
It is unclear how gene order within the chromosome influences genome evolution. Bacteria cluster transcription and translation genes close to the replication origin (oriC). In Vibrio cholerae, relocation of s10-spc-α locus (S10), the major locus of ribosomal protein genes, to ectopic genomic positions shows that its relative distance to the oriC correlates to a reduction in growth rate, fitness, and infectivity. To test the long-term impact of this trait, we evolved 12 populations of V. cholerae strains bearing S10 at an oriC-proximal or an oriC-distal location for 1,000 generations. During the first 250 generations, positive selection was the main force driving mutation. After 1,000 generations, we observed more nonadaptative mutations and hypermutator genotypes. Populations fixed inactivating mutations at many genes linked to virulence: flagellum, chemotaxis, biofilm, and quorum sensing. Throughout the experiment, all populations increased their growth rates. However, those bearing S10 close to oriC remained the fittest, indicating that suppressor mutations cannot compensate for the genomic position of the main ribosomal protein locus. Selection and sequencing of the fastest-growing clones allowed us to characterize mutations inactivating, among other sites, flagellum master regulators. Reintroduction of these mutations into the wild-type context led to a ≈10% growth improvement. In conclusion, the genomic location of ribosomal protein genes conditions the evolutionary trajectory of V. cholerae. While genomic content is highly plastic in prokaryotes, gene order is an underestimated factor that conditions cellular physiology and evolution. A lack of suppression enables artificial gene relocation as a tool for genetic circuit reprogramming. IMPORTANCE The bacterial chromosome harbors several entangled processes such as replication, transcription, DNA repair, and segregation. Replication begins bidirectionally at the replication origin (oriC) until the terminal region (ter) organizing the genome along the ori-ter axis gene order along this axis could link genome structure to cell physiology. Fast-growing bacteria cluster translation genes near oriC. In Vibrio cholerae, moving them away was feasible but at the cost of losing fitness and infectivity. Here, we evolved strains harboring ribosomal genes close or far from oriC. Growth rate differences persisted after 1,000 generations. No mutation was able to compensate for the growth defect, showing that ribosomal gene location conditions their evolutionary trajectory. Despite the high plasticity of bacterial genomes, evolution has sculpted gene order to optimize the ecological strategy of the microorganism. We observed growth rate improvement throughout the evolution experiment that occurred at expense of energetically costly processes such the flagellum biosynthesis and virulence-related functions. From the biotechnological point of view, manipulation of gene order enables altering bacterial growth with no escape events.
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Vibrio cholerae , Vibrio cholerae/genética , Proteínas Ribossômicas/genética , Genoma Bacteriano , Mutação , Cromossomos , Proteínas de Bactérias/genéticaRESUMO
Antimicrobial proteins and peptides are an alternative to current antibiotics. Here, we report an antimicrobial activity in a low-molecular-weight protein secreted naturally by Streptomyces lividans TK24 when glucose or glycerol were used as carbon sources. The antimicrobial activity was demonstrated against Bacillus subtilis, Bacillus cereus, Kokuria rhizophila, Clostridium sporogenes and Clavibacter michiganensis, causal pathogen of tomato bacterial canker; one of the most destructive bacterial diseases of this crop. The protein fraction with antimicrobial activity was identified and quantified by LC-MS/MS. From a total of 155 proteins, 11 were found to be within the range of 11.3-13.9 kDa of which four proteins were selected by functional analysis as possibly responsible for the antimicrobial activity. Protein fractionation, correlation analysis between antimicrobial activity and abundance of selected proteins, as well as transcriptional expression analysis, indicate that 50S ribosomal protein L19 is the main candidate responsible for antimicrobial activity.
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Anti-Infecciosos , Micrococcaceae , Solanum lycopersicum , Streptomyces lividans , Cromatografia Líquida , Espectrometria de Massas em Tandem , Solanum lycopersicum/microbiologia , Anti-Infecciosos/farmacologiaRESUMO
Moonlighting proteins are those capable of performing more than one biochemical or biophysical function within the same polypeptide chain. They have been a recent focus of research due to their potential applications in the health, pharmacological, and nutritional sciences. Among them, some ribosomal proteins involved in assembly and protein translation have also shown other functionalities, including inhibiting infectious bacteria, viruses, parasites, fungi, and tumor cells. Therefore, they may be considered antimicrobial peptides (AMPs). However, information regarding the mechanism of action of ribosomal proteins as AMPs is not yet fully understood. Researchers have suggested that the antimicrobial activity of ribosomal proteins may be associated with an increase in intracellular reactive oxidative species (ROS) in target cells, which, in turn, could affect membrane integrity and cause their inactivation and death. Moreover, the global overuse of antibiotics has resulted in an increase in pathogenic bacteria resistant to common antibiotics. Therefore, AMPs such as ribosomal proteins may have potential applications in the pharmaceutical and food industries in the place of antibiotics. This article provides an overview of the potential roles of ribosomes and AMP ribosomal proteins in conjunction with their potential applications.
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Anti-Infecciosos , Proteínas Ribossômicas , Antibacterianos/farmacologia , Anti-Infecciosos/farmacologia , Peptídeos Catiônicos Antimicrobianos/farmacologia , Bactérias , RibossomosRESUMO
OBJECTIVE: To systematically describe the short stature of patients with Diamond-Blackfan anemia and to explore factors affecting the height development of patients with Diamond-Blackfan anemia. STUDY DESIGN: This cross-sectional study was conducted at the Institute of Hematology and Blood Diseases Hospital, Chinese Academy of Medical Sciences and Peking Union Medical College, and the height, weight, and clinical data of 129 patients with Diamond-Blackfan anemia were collected from June 2020 to September 2020. RESULTS: The median height-age-z score (HAZ) of children affected by Diamond-Blackfan anemia was -1.54 (-6.36-1.96). Short stature was found in 37.98% of the patients. Specific Diamond-Blackfan anemia growth curves were developed for weight, height, and body mass index, separately for male and female patients. Multivariable logistic regression models showed that female sex (aOR 4.92; 95% CI 1.29-18.71; P = .0195), underweight (aOR 10.41, 95% CI 1.41-76.98, P = .0217), cardiovascular malformations (aOR 216.65; 95% CI 3.29-14279.79; P = .0118), and RPL11(aOR 29.14; 95% CI 1.18-719.10; P = .0392) or RPS26 (aOR 53.49; 95% CI 1.40-2044.30; P = .0323) mutations were independent risk factors for short stature. In the subgroup of patients who were steroid-dependent, patients with a duration of steroid therapy over 2 years (OR 2.95; 95% CI 1.00-8.66; P = .0494) or maintenance dose of prednisone >0.1 mg/kg per day (OR 3.30; 95% CI 1.02-10.72; P = .0470) had a higher incidence of short stature. CONCLUSIONS: Patients with Diamond-Blackfan anemia had a high prevalence of short stature. The risk of short stature increased with age and was associated with sex, underweight, congenital malformations, and RPL11 or RPS26 mutations. The duration of steroid therapy and maintenance dose of steroid was significantly associated with the incidence of short stature in steroid-dependent patients with Diamond-Blackfan anemia.
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Anemia de Diamond-Blackfan/epidemiologia , Nanismo/epidemiologia , Anormalidades Múltiplas/epidemiologia , Adolescente , Fatores Etários , Anemia de Diamond-Blackfan/tratamento farmacológico , Anemia de Diamond-Blackfan/genética , Criança , Pré-Escolar , China , Estudos Transversais , Nanismo/etiologia , Feminino , Glucocorticoides/administração & dosagem , Glucocorticoides/efeitos adversos , Humanos , Lactente , Masculino , Mutação , Prednisona/administração & dosagem , Prednisona/efeitos adversos , Proteínas Ribossômicas , Fatores SexuaisRESUMO
Ribosomal S6 kinase 1 (S6K1) and S6K2 proteins are effectors of the mammalian target of rapamycin complex 1 pathway, which control the process of protein synthesis in eukaryotes. S6K2 is associated with tumor progression and has a conserved C-terminus polyproline rich motif predicted to be important for S6K2 interactions. It is noteworthy that the translation of proteins containing sequential prolines has been proposed to be dependent of eukaryotic translation initiation factor 5A (eIF5A) translation factor. Therefore, we investigated the importance of polyproline-rich region of the S6K2 for its intrinsic phosphorylation activity, protein-protein interaction and eIF5A role in S6K2 translation. In HeLa cell line, replacing S6K2 polyproline by the homologous S6K1-sequence did not affect its kinase activity and the S6K2 endogenous content was maintained after eIF5A gene silencing, even after near complete depletion of eIF5A protein. Moreover, no changes in S6K2 transcript content was observed, ruling out the possibility of compensatory regulation by increasing the mRNA content. However, in the budding yeast model, we observed that S6K2 production was impaired when compared with S6K2∆Pro, after reduction of eIF5A protein content. These results suggest that although the polyproline region of S6K2 is capable of generating ribosomal stalling, the depletion of eIF5A in HeLa cells seems to be insufficient to cause an expressive decrease in the content of endogenous S6K2. Finally, coimmunoprecipitation assays revealed that the replacement of the polyproline motif of S6K2 alters its interactome and impairs its interaction with RPS6, a key modulator of ribosome activity. These results evidence the importance of S6K2 polyproline motif in the context of S6Ks function.
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Fatores de Iniciação de Peptídeos/química , Fatores de Iniciação de Peptídeos/metabolismo , Peptídeos/química , Isoformas de Proteínas/química , Isoformas de Proteínas/metabolismo , Proteínas de Ligação a RNA/química , Proteínas de Ligação a RNA/metabolismo , Proteínas Quinases S6 Ribossômicas/metabolismo , Inativação Gênica , Células HeLa , Humanos , Imunoprecipitação , Espectrometria de Massas , Fatores de Iniciação de Peptídeos/genética , Fosforilação , Reação em Cadeia da Polimerase , Ligação Proteica , Isoformas de Proteínas/genética , Proteínas de Ligação a RNA/genética , Proteínas Quinases S6 Ribossômicas/genética , Proteínas Quinases S6 Ribossômicas 70-kDa/genética , Proteínas Quinases S6 Ribossômicas 70-kDa/metabolismo , Fator de Iniciação de Tradução Eucariótico 5ARESUMO
Ribosomal S6 kinase 1 (S6K1) and S6K2 proteins are effectors of the mammalian target of rapamycin complex 1 pathway, which control the process of protein synthesis in eukaryotes. S6K2 is associated with tumor progression and has a conserved C-terminus polyproline rich motif predicted to be important for S6K2 interactions. It is noteworthy that the translation of proteins containing sequential prolines has been proposed to be dependent of eukaryotic translation initiation factor 5A (eIF5A) translation factor. Therefore, we investigated the importance of polyproline-rich region of the S6K2 for its intrinsic phosphorylation activity, protein-protein interaction and eIF5A role in S6K2 translation. In HeLa cell line, replacing S6K2 polyproline by the homologous S6K1-sequence did not affect its kinase activity and the S6K2 endogenous content was maintained after eIF5A gene silencing, even after near complete depletion of eIF5A protein. Moreover, no changes in S6K2 transcript content was observed, ruling out the possibility of compensatory regulation by increasing the mRNA content. However, in the budding yeast model, we observed that S6K2 production was impaired when compared with S6K2?Pro, after reduction of eIF5A protein content. These results suggest that although the polyproline region of S6K2 is capable of generating ribosomal stalling, the depletion of eIF5A in HeLa cells seems to be insufficient to cause an expressive decrease in the content of endogenous S6K2. Finally, coimmunoprecipitation assays revealed that the replacement of the polyproline motif of S6K2 alters its interactome and impairs its interaction with RPS6, a key modulator of ribosome activity. These results evidence the importance of S6K2 polyproline motif in the context of S6Ks function.
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To characterize the structure and function of ribosomal protein S13 (RPS13), we identified fulllength open reading frames (ORFs) of three RPS13 genes (RPS13-1, RPS13-2, and RPS13-3) of the Chinese medicinal plant, Sophora flavescens. The target genes were amplified by reverse transcription-olymerase chain reaction (RT-PCR), ligated into the pET22b(+) vector, and then transformed into Escherichia coli BL21 competent cells for protein expression. The physicochemical properties, protein motif, evolution, and structural organization of the three RPS13 genes were analyzed using bioinformatics tools. The full-length ORFs (453 bp) of the three RPS13 genes of S. flavescens were cloned, and each encodes a protein of 151 amino acids in length, and their expression was detected by Western blotting. Bioinformatics analysis showed that RPS13s are stable proteins that are closely related to the 40S RPS13s of Vigna radiate var. radiate. Their three-dimensional structures included three -helices at the C-terminal and four -helices at the N-terminal, and the two clusters of helices were connected by a long random coil, which may help maintain the dynamic bridging interactions between the large and small subunits of the ribosome. The full-length ORFs of three RPS13 genes of S. flavescens were successfully cloned and expressed in vitro. The study of the physicochemical properties, evolution, and secondary and three-dimensional structures of the three proteins will provide the theoretical basis for further studies on the function of RPS13s in plants.
Objetivo: Para caracterizar a estrutura e a função da proteína ribossomal S13 (RPS13), identificamos fases de leitura abertas (ORFs) completas de três genes RPS13 (RPS13-1, RPS13-2 e RPS13-3) da planta medicinal chinesa, Sophora flavescens. Métodos: Os genes alvo foram amplificados por reação em cadeia da polimerase por transcrição reversa (RT-PCR), ligados ao vetor pET22b(+), e então transformados em células competentes de Escherichia coli BL21 para expressão protéica. As propriedades físico-químicas, o motivo protéico, a evolução e a organização estrutural dos três genes RPS13 foram analisados utilizando ferramentas de bioinformática. Resultados: ORFs completos (453 pb) dos três genes RPS13 de S. flavescens foram clonados, e cada um codifica uma proteína de 151 aminoácidos de comprimento, e sua expressão foi detectada por western blotting. A análise de bioinformática mostrou que as RPS13s são proteínas estáveis que estão intimamente relacionadas com as 40S RPS13s de Vigna radiata var. radiate. Suas estruturas tridimensionais incluíam três -hélices no C-terminal e quatro -hélices no N-terminal, e os dois aglomerados de hélices eram conectados por uma longa bobina aleatória, o que pode ajudar a manter as interações de ponte dinâmicas entre o subunidades grandes e pequenas do ribossomo. Conclusões: As ORFs completas de três genes RPS13 de S. flavescens foram clonadas e expressas com sucesso in vitro. O estudo das propriedades físico-químicas, evolução e estruturas secundárias e tridimensionais das três proteínas fornecerão a base teórica para estudos adicionais sobre a função das RPS13s em plantas.
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Biologia Computacional , Sophora , Transcrição Reversa , Escherichia coli , GenesRESUMO
The LrtA protein of Synechocystis sp. PCC 6803 intervenes in cyanobacterial post-stress survival and in stabilizing 70S ribosomal particles. It belongs to the hibernating promoting factor (HPF) family of proteins, involved in protein synthesis. In this work, we studied the conformational preferences and stability of isolated LrtA in solution. At physiological conditions, as shown by hydrodynamic techniques, LrtA was involved in a self-association equilibrium. As indicated by Nuclear Magnetic Resonance (NMR), circular dichroism (CD) and fluorescence, the protein acquired a folded, native-like conformation between pH 6.0 and 9.0. However, that conformation was not very stable, as suggested by thermal and chemical denaturations followed by CD and fluorescence. Theoretical studies of its highly-charged sequence suggest that LrtA had a Janus sequence, with a context-dependent fold. Our modelling and molecular dynamics (MD) simulations indicate that the protein adopted the same fold observed in other members of the HPF family (ß-α-ß-ß-ß-α) at its N-terminal region (residues 1100), whereas the C terminus (residues 100197) appeared disordered and collapsed, supporting the overall percentage of overall secondary structure obtained by CD deconvolution. Then, LrtA has a chameleonic sequence and it is the first member of the HPF family involved in a self-association equilibrium, when isolated in solution.
Assuntos
Proteínas de Bactérias/química , Proteínas Ribossômicas/química , Ribossomos/química , Synechocystis/química , Motivos de Aminoácidos , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Clonagem Molecular , Escherichia coli/genética , Escherichia coli/metabolismo , Expressão Gênica , Vetores Genéticos/química , Vetores Genéticos/metabolismo , Simulação de Dinâmica Molecular , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Dobramento de Proteína , Domínios e Motivos de Interação entre Proteínas , Multimerização Proteica , Estabilidade Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Proteínas Ribossômicas/genética , Proteínas Ribossômicas/metabolismo , Ribossomos/metabolismo , Soluções , Synechocystis/metabolismo , TermodinâmicaRESUMO
PI3K/Akt/mTOR pathway activation is a hallmark of high-grade gliomas, which prompted clinical trials for the use of PI3K and mTOR inhibitors. However, the poor results in the original trials suggested that better patient profiling was needed for such drugs. Thus, accurate and reproducible monitoring of mTOR complexes can lead to improved therapeutic strategies. In this work, we evaluated the expression and phosphorylation of mTOR, RAPTOR, and rpS6 in 195 human astrocytomas and 30 normal brain tissue samples. The expression of mTOR increased in glioblastomas, whereas mTOR phosphorylation, expression of RAPTOR, and expression and phosphorylation of rpS6 were similar between grades. Interestingly, the overexpression of total and phosphorylated mTOR as well as phosphorylated rpS6 (residues 240-244) were associated with wild-type IDH1 only glioblastomas. The expression and phosphorylation of mTOR and phosphorylation of rpS6 at residues 240-244 were associated with a worse prognosis in glioblastomas. Our results suggest that mTOR and rpS6 could be used as markers of overactivation of the PI3K-mTOR pathway and are predictive factors for overall survival in glioblastomas. Our study thus suggests that patients who harbor IDH1 wild-type glioblastomas might have increased benefit from targeted therapy against mTOR.
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Neoplasias Encefálicas/patologia , Glioblastoma/patologia , Isocitrato Desidrogenase/análise , Proteínas Quinases S6 Ribossômicas/análise , Serina-Treonina Quinases TOR/análise , Regulação para Cima , Adulto , Neoplasias Encefálicas/diagnóstico , Neoplasias Encefálicas/epidemiologia , Criança , Feminino , Glioblastoma/diagnóstico , Glioblastoma/epidemiologia , Humanos , Imuno-Histoquímica/métodos , Masculino , Pessoa de Meia-Idade , Fosforilação , Prognóstico , Taxa de SobrevidaRESUMO
In Schizosaccharomyces pombe, ribosomal protein gene (RPG) promoters contain a TATA box analog, the HomolD box, which is bound by the Rrn7 protein. Despite the importance of ribosome biogenesis for cell survival, the mechanisms underlying RPG transcription remain unknown. In this study, we found that components of the RNA polymerase II (RNAPII) system, consisting of the initiation or general transcription factors (GTFs) TFIIA, IIB, IIE, TATA-binding protein (TBP) and the RNAPII holoenzyme, interacted directly with Rrn7 in vitro, and were able to form a preinitiation complex (PIC) on the HomolD box. PIC complex formation follows an ordered pathway on these promoters. The GTFs and RNAPII can also be cross-linked to HomolD-containing promoters in vivo. In an in vitro reconstituted transcription system, RNAPII components and Rrn7 were necessary for HomolD-directed transcription. The Mediator complex was required for basal transcription from those promoters in whole cell extract (WCE). The Med17 subunit of Mediator also can be cross-linked to the promoter region of HomolD-containing promoters in vivo, suggesting the presence of the Mediator complex on HomolD box-containing promoters. Together, these data show that components of the RNAPII machinery and Rrn7 participate in the PIC assembly on the HomolD box, thereby directing RPG transcription.
Assuntos
Proteínas Fúngicas/genética , Regulação Fúngica da Expressão Gênica , Proteínas Pol1 do Complexo de Iniciação de Transcrição/genética , Proteínas Ribossômicas/genética , Schizosaccharomyces/genética , TATA Box , Sítios de Ligação , Clonagem Molecular , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas Fúngicas/metabolismo , Expressão Gênica , Complexo Mediador/genética , Complexo Mediador/metabolismo , Proteínas Pol1 do Complexo de Iniciação de Transcrição/metabolismo , Regiões Promotoras Genéticas , Ligação Proteica , RNA Polimerase II/genética , RNA Polimerase II/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Proteínas Ribossômicas/metabolismo , Schizosaccharomyces/metabolismo , Proteína de Ligação a TATA-Box/genética , Proteína de Ligação a TATA-Box/metabolismo , Fator de Transcrição TFIIA/genética , Fator de Transcrição TFIIA/metabolismo , Fator de Transcrição TFIIB/genética , Fator de Transcrição TFIIB/metabolismo , Fatores de Transcrição TFII/genética , Fatores de Transcrição TFII/metabolismo , Transcrição GênicaRESUMO
In Schizosaccharomyces pombe, ribosomal protein gene (RPG) promoters contain a TATA analogue element called the HomolD box. The HomolD-binding protein Rrn7 forms a complex with the RNA polymerase II machinery. Despite the importance of ribosome biogenesis to cell survival, the mechanisms involved in the regulation of transcription of eukaryotic RPGs are unknown. In this study, we identified Rrn7 as a new substrate of the pleiotropic casein kinase 2 (CK2), which is a regulator of basal transcription. Recombinant Rrn7 from S. pombe, which is often used as a model organism for studying eukaryotic transcription, interacted with CK2 in vitro and in vivo. Furthermore, CK2-mediated phosphorylation of Rrn7 inhibited its HomolD-directed transcriptional activity and ability to bind to an oligonucleotide containing a HomolD box in vitro. Mutation of Rrn7 at Thr67 abolished these effects, indicating that this residue is a critical CK2 phosphorylation site. Finally, Rrn7 interacted with the regulatory subunit of CK2 in vivo, inhibition of CK2 in vivo potentiated ribosomal protein gene transcription, and chromatin immunoprecipitation analyses identified that the catalytic subunit of CK2 was associated with the rpk5 gene promoter in S. pombe. Taken together, these data suggest that CK2 inhibits ribosomal protein gene transcription in S. pombe via phosphorylation of Rrn7 at Thr67.
Assuntos
Caseína Quinase II/metabolismo , Proteínas Pol1 do Complexo de Iniciação de Transcrição/metabolismo , Proteínas de Schizosaccharomyces pombe/metabolismo , Schizosaccharomyces/enzimologia , Schizosaccharomyces/metabolismo , Fosforilação , Ligação ProteicaRESUMO
In this Extra View we comment on our recent work on Sudestada1 (Sud1), a Drosophila 2-oxoglutarate (2OG)-dependent dioxygenase that belongs to the Ribosomal Oxygenase (ROX) subfamily. Sud1 is required for normal growth in Drosophila, and is conserved in yeast and mammals. We reported that Sud1 hydroxylates the ribosomal protein S23 (RPS23), and that its loss of function restricts growth and provokes activation of the unfolded protein response, apoptosis and autophagy. In this Extra View we speculate on the role that RPS23 hydroxylation might play in stop codon recognition and on the possible link between Sud1 loss-of-function and activation of the Unfolded Protein Response, Stress Granules formation and growth impairment.
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Processos de Crescimento Celular , Proteínas de Drosophila/metabolismo , Drosophila/enzimologia , Regulação da Expressão Gênica , Prolil Hidroxilases/metabolismo , Proteínas Ribossômicas/metabolismo , Animais , HidroxilaçãoRESUMO
Leishmania is a genus of protozoan parasites causing a wide clinical spectrum of diseases in humans. Analysis of a region of chromosome 6 from Leishmania major (Iribar et al.) showed that the transcript of a putative L19 ribosomal protein (RPL19) was most abundant at the amastigote stage. We therefore decided to characterize L19 protein abundance throughout the lifecycle of Leishmania. Differential expression of the L19 gene during development has been observed for all Leishmania species studied to date (L. major, L. braziliensis, L. donovani, and L. amazonensis). Immunoblotting with polyclonal antibodies against L. major RPL19 revealed that changes to L19 protein abundance follow a similar pattern in various species. The amount of L19 protein was higher in exponentially growing promastigotes than in stationary phase promastigotes. The L19 protein was barely detectable in amastigotes, despite the abundance of L19 transcripts observed in L. major at this stage. Immunofluorescence assays showed a granular, dispersed distribution of RPL19 throughout the cytoplasm. Subcellular fractionation confirmed the presence of the protein in the ribosomal fraction, but not in the cytosol of L. major. We generated a L. major transfectant bearing a plasmid-borne L19 gene. Overproduction of the L19 transcript and protein resulted in impaired growth of the transfectants in association with high polysome peaks. We also showed by metabolic labeling that L19 overexpressing clones display low rates of translation. These data suggest that L19 overexpression affects negatively translation elongation or termination. The lack of correlation between L19 transcript and protein abundances suggest that the translation of L19 is differentially controlled during development in the various species investigated.
Assuntos
Regulação da Expressão Gênica no Desenvolvimento/fisiologia , Leishmania/crescimento & desenvolvimento , Leishmaniose/parasitologia , Estágios do Ciclo de Vida/fisiologia , Proteínas Ribossômicas/metabolismo , Animais , Anticorpos Antiprotozoários/biossíntese , Anticorpos Antiprotozoários/imunologia , Northern Blotting , Western Blotting , Clonagem Molecular , Feminino , Imunofluorescência , Regulação da Expressão Gênica no Desenvolvimento/genética , Vetores Genéticos , Leishmania/genética , Leishmania/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Polirribossomos/química , RNA Mensageiro/análise , RNA Mensageiro/isolamento & purificação , RNA de Protozoário/análise , RNA de Protozoário/isolamento & purificação , Coelhos , Proteínas Ribossômicas/genética , Proteínas Ribossômicas/imunologia , Ribossomos/genéticaRESUMO
BACKGROUND: Type 2 diabetes (T2D) and heart failure (HF) are associated with high levels of skeletal muscle (SkM) oxidative stress (OS). Health benefits attributed to flavonoids have been ascribed to antioxidation. However, for flavonoids with similar antioxidant potential, end-biological effects vary widely suggesting other mechanistic venues for reducing OS. Decreases in OS may follow the modulation of key regulatory pathways including antioxidant levels (e.g. glutathione) and enzymes such as mitochondrial superoxide dismutase (SOD2) and catalase. METHODS: We examined OS-related alterations in SkM in T2D/HF patients (as compared vs. healthy controls) and evaluated the effects of three-month treatment with (-)-epicatechin (Epi) rich cocoa (ERC). To evidence Epi as the mediator of the improved OS profile we examined the effects of pure Epi (vs. water) on SkM OS regulatory systems in a mouse model of insulin resistance and contrasted results vs. normal mice. RESULTS: There were severe alterations in OS regulatory systems in T2D/HF SkM as compared with healthy controls. Treatment with ERC induced recovery in glutathione levels and decreases in the nitrotyrosilation and carbonylation of proteins. With treatment, key transcriptional factors translocate into the nucleus leading to increases in SOD2 and catalase protein expression and activity levels. In insulin resistant mice, there were alterations in muscle OS and pure Epi replicated the beneficial effects of ERC found in humans. CONCLUSIONS: Major perturbations in SkM OS can be reversed with ERC in T2D/HF patients. Epi likely mediates such effects and may provide an effective means to treat conditions associated with tissue OS.
Assuntos
Cacau , Catequina/administração & dosagem , Diabetes Mellitus Tipo 2/metabolismo , Insuficiência Cardíaca/metabolismo , Músculo Esquelético/metabolismo , Estresse Oxidativo/fisiologia , Idoso , Animais , Bebidas , Diabetes Mellitus Tipo 2/tratamento farmacológico , Feminino , Insuficiência Cardíaca/tratamento farmacológico , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Pessoa de Meia-Idade , Músculo Esquelético/efeitos dos fármacos , Estresse Oxidativo/efeitos dos fármacos , Projetos PilotoRESUMO
Ribosome biogenesis is a pre-requisite for cell growth and proliferation; it is however, a highly regulated process that consumes a great quantity of energy. It requires the coordinated production of rRNA, ribosomal proteins and non-ribosomal factors which participate in the processing and mobilization of the new ribosomes. Ribosome biogenesis has been studied in yeast and animals; however, there is little information about this process in plants. The objective of the present work was to study ribosome biogenesis in maize seeds during germination, a stage characterized for its fast growth, and the effect of insulin in this process. Insulin has been reported to accelerate germination and to induce seedling growth. It was observed that among the first events reactivated just after 3 h of imbibition are the rDNA transcription and the pre-rRNA processing and that insulin stimulates both of them (40-230%). The transcript of nucleolin, a protein which regulates rDNA transcription and pre-rRNA processing, is among the messages stored in quiescent dry seeds and it is mobilized into the polysomal fraction during the first hours of imbibition (6 h). In contrast, de novo ribosomal protein synthesis was low during the first hours of imbibition (3 and 6 h) increasing by 60 times in later stages (24 h). Insulin increased this synthesis (75%) at 24 h of imbibition; however, not all ribosomal proteins were similarly regulated. In this regard, an increase in RPS6 and RPL7 protein levels was observed, whereas RPL3 protein levels did not change even though its transcription was induced. Results show that ribosome biogenesis in the first stages of imbibition is carried out with newly synthesized rRNA and ribosomal proteins translated from stored mRNA.
Assuntos
DNA Ribossômico/genética , Regulação da Expressão Gênica de Plantas , Germinação/genética , Ribossomos/genética , Sementes/genética , Zea mays/genética , DNA Ribossômico/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Insulina/farmacologia , Fosfoproteínas/genética , Fosfoproteínas/metabolismo , Processamento Pós-Transcricional do RNA , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo , Proteínas Ribossômicas/genética , Proteínas Ribossômicas/metabolismo , Ribossomos/efeitos dos fármacos , Ribossomos/metabolismo , Sementes/efeitos dos fármacos , Sementes/crescimento & desenvolvimento , Sementes/metabolismo , Fatores de Tempo , Transcrição Gênica , Zea mays/efeitos dos fármacos , Zea mays/crescimento & desenvolvimento , Zea mays/metabolismo , NucleolinaRESUMO
Chronic atrophic gastritis (CAG) is a very common gastritis and one of the major precursor lesions of gastric cancer, one of the most common cancers worldwide. The molecular mechanism underlying CAG is unclear, but its elucidation is essential for the prevention and early detection of gastric cancer and appropriate intervention. A combination of two-dimensional gel electrophoresis and mass spectrometry was used in the present study to analyze the differentially expressed proteins. Samples from 21 patients (9 females and 12 males; mean age: 61.8 years) were used. We identified 18 differentially expressed proteins in CAG compared with matched normal mucosa. Eight proteins were up-regulated and 10 down-regulated in CAG when compared with the same amounts of proteins in individually matched normal gastric mucosa. Two novel proteins, proteasome activator subunit 1 (PSME1), which was down-regulated in CAG, and ribosomal protein S12 (RPS12), which was up-regulated in CAG, were further investigated. Their expression was validated by Western blot and RT-PCR in 15 CAG samples matched with normal mucosa. The expression level of RPS12 was significantly higher in CAG than in matched normal gastric mucosa (P < 0.05). In contrast, the expression level of PSME1 in CAG was significantly lower than in matched normal gastric mucosa (P < 0.05). This study clearly demonstrated that there are some changes in protein expression between CAG and normal mucosa. In these changes, down-regulation of PSME1 and up-regulation of RPS12 could be involved in the development of CAG. Thus, the differentially expressed proteins might play important roles in CAG as functional molecules.
Assuntos
Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Mucosa Gástrica/química , Gastrite Atrófica/metabolismo , Proteínas Musculares/genética , Proteômica , Complexo de Endopeptidases do Proteassoma/genética , Proteínas Ribossômicas/metabolismo , Western Blotting , Doença Crônica , Regulação para Baixo , Eletroforese em Gel Bidimensional , Mucosa Gástrica/patologia , Gastrite Atrófica/genética , Helicobacter pylori , Espectrometria de Massas , Proteínas Musculares/metabolismo , Complexo de Endopeptidases do Proteassoma/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Proteínas Ribossômicas/genética , Regulação para CimaRESUMO
This study was undertaken to characterize the alpha subgroup of the proteobacteria causing the huanglongbing (HLB) disease of citrus from three different ecological zones of Kenya namely the Lower highlands (LH2, LH3, 1800-1900 m above sea level); Upper midlands (UM3, UM4, 1390-1475m), Lower midlands (LM5, LM4, LM3 of 1290-1340-1390m), by isolation and sequencing DNA encoding the L10 and L12 ribosomal proteins and the intergenic region. A 7I6-basepair DNA fragment was amplified and sequenced and consisted of 536 basepairs of DNA encoding the L10 protein, 44 basepairs of DNA intergenic region and 136 basepairs of DNA that partially encodes the L12 protein. Sequences of rpL10/L12 protein genes from Kenyan strains were 98 percent and 81 percent similar to the South African 'Candidatus Liberibacter africanus strain Nelspruit' and the Asian 'Candidatus Liberibacter asiaticus' strains, respectively. The intergenic rDNA sequence of Kenyan strain from UM and LM showed 84 percent similarity with 'Candidatus L. africanus strain Nelspruit' and 50 percent similarity with 'Candidatus L. asiaticus' strain. However, the LH strain had an 11- basepairs deletion, while the LM4 had a 5-basepair deletion in the intergenic region compared to 'Candidatus L. africanus strain Nelspruit'. The L10 amino acid sequence was 100 percent homologous among HLB bacteria obtained from the agro-ecological zones in Kenya and the L10 protein sequence was also homologus to 'Candidatus L. africanus strain Nelspruit'. Nevertheless, the L10 amino acid sequence of 'Candidatus L. asiaticus' and the 'Candidatus L. africanus subsp. capensis' differed from the Kenyan strains by 18.36 percent and 11.82 percent, respectively. Phylogenetic analysis of both the L10/L12 rDNA sequences and the L10 amino acid sequences clustered the Kenyan strains of the 'Candidatus Liberibacter' species with members of alpha subdivision of proteobacteria.