RESUMO
Emerging evidence suggests that ribosome heterogeneity may have important functional consequences in the translation of specific mRNAs within different cell types and under various conditions. Ribosome heterogeneity comes in many forms, including post-translational modification of ribosome proteins (RPs), absence of specific RPs and inclusion of different RP paralogs. The Drosophila genome encodes two RpS5 paralogs: RpS5a and RpS5b. While RpS5a is ubiquitously expressed, RpS5b exhibits enriched expression in the reproductive system. Deletion of RpS5b results in female sterility marked by developmental arrest of egg chambers at stages 7-8, disruption of vitellogenesis and posterior follicle cell (PFC) hyperplasia. While transgenic rescue experiments suggest functional redundancy between RpS5a and RpS5b, molecular, biochemical and ribo-seq experiments indicate that RpS5b mutants display increased rRNA transcription and RP production, accompanied by increased protein synthesis. Loss of RpS5b results in microtubule-based defects and in mislocalization of Delta and Mindbomb1, leading to failure of Notch pathway activation in PFCs. Together, our results indicate that germ cell-specific expression of RpS5b promotes proper egg chamber development by ensuring the homeostasis of functional ribosomes.
Assuntos
Infertilidade/genética , Oogênese , Oogônios/metabolismo , Folículo Ovariano/metabolismo , Animais , Proteínas de Drosophila/metabolismo , Drosophila melanogaster , Feminino , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteínas de Membrana/metabolismo , Mutação , Oogônios/citologia , Folículo Ovariano/citologia , Transporte Proteico , RNA Ribossômico/genética , RNA Ribossômico/metabolismo , Receptores Notch/metabolismo , Transdução de SinaisRESUMO
Hypoxia-induced vasoconstriction and vascular remodelling are the main pathological features of hypoxic pulmonary arterial hypertension (HPAH), and inflammation is participated in the occurrence of pulmonary vascular remodelling (PVR). Matrine is an alkaloid with the effects of anti-inflammation, antifibrosis and antitumour. But, few studies have explored the role of matrine in regulating PVR, and the related mechanisms are still unknown. In this study, we found that hypoxia-induced pulmonary artery smooth muscle cells (PASMCs) proliferation and inhibited its apoptosis, reduced the expression of ribosomal protein s5 and activated the nuclear factor kappa-B (NF-κB) signalling. Matrine, sildenafil and NF-κB inhibitor Bay 11-7082 could reverse these changes and impel the cell cycle in phase S retardation, and reduced the expression of p50, p65, proliferating cell nuclear antigen (PCNA), Bcl-2. In addition, matrine could lower right ventricular systolic pressure and mean pulmonary artery pressure of rats, α-smooth muscle actin and PCNA expression in pulmonary artery media, the levels of tumor necrosis factor-α and interleuki-1ß, thus improved hypoxia-induced PVR. This study indicated that matrine could alleviate inflammation and improve PVR through reversing the imbalance of proliferation and apoptosis of PASMCs, thus it had a therapeutic effect on HPAH.
Assuntos
Hidralazina/análogos & derivados , Hipertensão Pulmonar , NF-kappa B , Ratos , Animais , NF-kappa B/metabolismo , Antígeno Nuclear de Célula em Proliferação/metabolismo , Matrinas , Hipertensão Pulmonar/tratamento farmacológico , Hipertensão Pulmonar/etiologia , Hipertensão Pulmonar/patologia , Remodelação Vascular , Proliferação de Células , Hipóxia/complicações , Hipóxia/metabolismo , Inflamação/metabolismo , Miócitos de Músculo Liso/metabolismo , HidrazonasRESUMO
Hepatocellular carcinoma (HCC) represents a major global health concern, demanding a thorough understanding of its molecular mechanisms for effective therapeutic strategies. RNA-binding proteins (RBPs) play critical roles in post-transcriptional gene regulation, with their dysregulation increasingly recognized as a hallmark of various cancers. However, the specific contributions of RBPs to HCC pathogenesis and prevention remain incompletely understood. In this study, we systematically conducted an examination of the expression profiles and clinical relevance of RBPs in 556 clinical samples from well-established cohorts. Through comprehensive analyses, a subset of RBPs exhibiting significant overexpression in HCC was identified, establishing a noteworthy correlation between their aberrant expression and HCC progression. Furthermore, 40S ribosomal protein S5 (RPS5), a ribosomal protein, emerged as a potential key contributor in HCC progression. Rigorous analyses established a correlation between elevated RPS5 expression and advanced clinicopathological features, suggesting its potential as a prognostic biomarker. Experiments further confirmed the impact of RPS5 on pivotal cellular processes implicated in cancer progression, including cell proliferation and metastasis. Further mechanistic studies unveiled the potential of RPS5 to activate the cell cycle by binding to key molecules involved in the pathway, thereby promoting the malignant progression of HCC. Additionally, our analysis of the etiology behind RPS5 overexpression in HCC posited it as an outcome of transcriptional regulation by the transcription factors Nuclear Respiratory Factor 1 (NRF1) and MYC-associated zinc finger protein (MAZ). In conclusion, our study contributes to the growing evidence elucidating the intricate involvement of RBPs, exemplified by RPS5, in the malignant progression of HCC. The integration of genomic, transcriptomic, and functional analyses provides a comprehensive understanding of the regulatory mechanisms associated with RPS5 in HCC. This comprehensive analysis not only advances our knowledge of the molecular drivers behind HCC but also highlights the potential therapeutic relevance of targeting RBPs and their regulatory network for the development of more effective treatment strategies.
Assuntos
Carcinoma Hepatocelular , Neoplasias Hepáticas , Humanos , Carcinoma Hepatocelular/genética , Neoplasias Hepáticas/genética , Proteínas Ribossômicas/genética , Proteínas de Ligação a RNA/genéticaRESUMO
OBJECTIVES: Stem cells of the apical papilla (SCAPs) provide promising candidates for dental pulp regeneration. Despite great advances in the transcriptional controls of the SCAPs fate, little is known about the regulation of SCAP differentiation. MATERIALS AND METHODS: Short hairpin RNAs and full-length RNA were used to deplete or overexpress lysine demethylase 4D (KDM4D) gene expression. Western blotting, real-time RT-PCR, alizarin red staining, and scratch migration assays were used to study the role of KDM4D and the ribosomal protein encoded by RPS5 in SCAPs. RNA microarray, chromatin Immunoprecipitation (ChIP), and co-immunoprecipitation (Co-IP) assays were performed to explore the underlying molecular mechanisms. RESULTS: KDM4D enhanced the osteo/dentinogenic differentiation, migration, and chemotaxis of SCAPs. The microarray results revealed that 88 mRNAs were differentially expressed in KDM4D-overexpressed SCAPs. ChIP results showed knock-down of KDM4D increased the level of H3K9me2 and H3K9me3 in CNR1 promoter region. There were 37 possible binding partners of KDM4D. KDM4D was found to combine with RPS5, which also promoted the osteo/dentinogenic differentiation, migration, and chemotaxis of SCAPs. CONCLUSIONS: KDM4D promoted the osteo/dentinogenic differentiation and migration potential of SCAPs in combination with RPS5, which provides a therapeutic clue for improving SCAPs-based dental tissue regeneration.
Assuntos
Polpa Dentária , Histona Desmetilases com o Domínio Jumonji , Regeneração , Diferenciação Celular , Proliferação de Células , Células Cultivadas , Papila Dentária/metabolismo , Polpa Dentária/metabolismo , Osteogênese/genética , RNA Interferente Pequeno , Células-Tronco , Humanos , Histona Desmetilases com o Domínio Jumonji/genéticaRESUMO
Nucleotide-binding site (NBS)-leucine-rich repeat (LRR) domain receptor (NLR) proteins play important roles in plant innate immunity by recognizing pathogen effectors. The Toll/interleukin-1 receptor (TIR)-NBS (TN) proteins belong to a subtype of the atypical NLRs, but their function in plant immunity is poorly understood. The well-characterized Arabidopsis thaliana typical coiled-coil (CC)-NBS-LRR (CNL) protein Resistance to Pseudomonas syringae 5 (RPS5) is activated after recognizing the Pseudomonas syringae type III effector AvrPphB. To explore whether the truncated TN proteins function in CNL-mediated immune signaling, we examined the interactions between the Arabidopsis TN proteins and RPS5, and found that TN13 and TN21 interacted with RPS5. However, only TN13, but not TN21, was involved in the resistance to P. syringae pv. tomato (Pto) strain DC3000 carrying avrPphB, encoding the cognate effector recognized by RPS5. Moreover, the regulation of Pto DC3000 avrPphB resistance by TN13 appeared to be specific, as loss of function of TN13 did not compromise resistance to Pto DC3000 hrcC- or Pto DC3000 avrRpt2. In addition, we demonstrated that the CC and NBS domains of RPS5 play essential roles in the interaction between TN13 and RPS5. Taken together, our results uncover a direct functional link between TN13 and RPS5, suggesting that TN13 acts as a partner in modulating RPS5-activated immune signaling, which constitutes a previously unknown mechanism for TN-mediated regulation of plant immunity.
Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Regulação da Expressão Gênica de Plantas/imunologia , Proteínas de Membrana/metabolismo , Doenças das Plantas/imunologia , Arabidopsis/microbiologia , Proteínas de Arabidopsis/genética , Proteínas de Membrana/genética , Doenças das Plantas/genética , Doenças das Plantas/microbiologia , Pseudomonas syringaeRESUMO
The AVRPPHB SUSCEPTIBLE1 (PBS1) and RESISTANCE TO PSEUDOMONAS SYRINGAE 5 (RPS5) proteins are involved in signal transduction to evoke innate plant immune response. In Arabidopsis, PBS1 is cleaved by the AvrPphB (Pseudomonas phaseolicola Avirulence protein B) protease, activating RPS5 and turning in a hypersensitive response (HR). We searched for PBS1 orthologs to trace their origin and evolution. PBS1 orthologs were found in embryophytes and in other plant taxa but with lower similarity. PBS1 phylogenetic analysis indicates high divergence, suggesting that the decoy function described for Arabidopsis PBS1 might be associated with a small fraction of orthologs. Ancestral reconstruction analysis suggests an elevated diversity in the amino acid sequence within the described motifs. All the orthologs contain the conserved PBS1 kinase subdomains, whereas the cleavage motif is present in several embryophyte orthologs but absent in most other taxa. The putative resistance recognition motifs in PBS1 orthologs are highly diverse. PBS1 cleavage site motif is exposed in some 3D structure predictions, whereas it is not in others, suggesting different modes of regulation and functions in PBS1 orthologs. Our findings suggest that PBS1 originated in the lineage that gave rise to embryophytes, with the angiosperm sequences forming a separate clade from pteridophyte proteins.
Assuntos
Evolução Biológica , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Plantas/genética , Plantas/metabolismo , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , Sequência de Aminoácidos , Sequência Conservada , Regulação da Expressão Gênica de Plantas , Modelos Moleculares , Filogenia , Fenômenos Fisiológicos Vegetais , Conformação Proteica , Proteínas Serina-Treonina Quinases/química , Transdução de Sinais , Relação Estrutura-AtividadeRESUMO
The Arabidopsis resistance protein RPS5 is activated by proteolytic cleavage of the protein kinase PBS1 by the Pseudomonas syringae effector protease AvrPphB. We have previously shown that replacing seven amino acids at the cleavage site of PBS1 with a motif cleaved by the NIa protease of turnip mosaic virus (TuMV) enables RPS5 activation upon TuMV infection. However, this engineered resistance conferred a trailing necrosis phenotype indicative of a cell-death response too slow to contain the virus. We theorized this could result from a positional mismatch within the cell between PBS1TuMV, RPS5, and the NIa protease. To test this, we relocalized PBS1TuMV and RPS5 to cellular sites of NIa accumulation. These experiments revealed that relocation of RPS5 away from the plasma membrane compromised RPS5-dependent cell death in Nicotiana benthamiana, even though PBS1 was efficiently cleaved. As an alternative approach, we tested whether overexpression of plasma membrane-localized PBS1TuMV could enhance RPS5 activation by TuMV. Significantly, overexpressing the PBS1TuMV decoy protein conferred complete resistance to TuMV when delivered by either agrobacterium or by aphid transmission, showing that RPS5-mediated defense responses are effective against bacterial and viral pathogens. Lastly, we have now extended this PBS1 decoy approach to soybean by modifying a soybean PBS1 ortholog to be cleaved by the NIa protease of soybean mosaic virus (SMV). Transgenic overexpression of this soybean PBS1 decoy conferred immunity to SMV, demonstrating that we can use endogenous PBS1 proteins in crop plants to engineer economically relevant disease resistant traits.
Assuntos
Proteínas de Arabidopsis/genética , Arabidopsis/virologia , Resistência à Doença/genética , Glycine max/virologia , Doenças das Plantas/virologia , Potyvirus/patogenicidade , Proteínas Serina-Treonina Quinases/genética , Animais , Arabidopsis/genética , Plantas Geneticamente Modificadas/virologia , Glycine max/genéticaRESUMO
Genetic resources for the model plant Arabidopsis comprise mutant lines defective in almost any single gene in reference accession Columbia. However, gene redundancy and/or close linkage often render it extremely laborious or even impossible to isolate a desired line lacking a specific function or set of genes from segregating populations. Therefore, we here evaluated strategies and efficiencies for the inactivation of multiple genes by Cas9-based nucleases and multiplexing. In first attempts, we succeeded in isolating a mutant line carrying a 70 kb deletion, which occurred at a frequency of ~ 1.6% in the T2 generation, through PCR-based screening of numerous individuals. However, we failed to isolate a line lacking Lhcb1 genes, which are present in five copies organized at two loci in the Arabidopsis genome. To improve efficiency of our Cas9-based nuclease system, regulatory sequences controlling Cas9 expression levels and timing were systematically compared. Indeed, use of DD45 and RPS5a promoters improved efficiency of our genome editing system by approximately 25-30-fold in comparison to the previous ubiquitin promoter. Using an optimized genome editing system with RPS5a promoter-driven Cas9, putatively quintuple mutant lines lacking detectable amounts of Lhcb1 protein represented approximately 30% of T1 transformants. These results show how improved genome editing systems facilitate the isolation of complex mutant alleles, previously considered impossible to generate, at high frequency even in a single (T1) generation.
Assuntos
Arabidopsis/genética , Proteína 9 Associada à CRISPR/genética , Edição de Genes/métodos , Alelos , Proteína 9 Associada à CRISPR/metabolismo , Sistemas CRISPR-Cas , Deleção de Genes , Genoma de Planta , Mutação , Regiões Promotoras Genéticas , Ubiquitina/genéticaRESUMO
BACKGROUND: Reference genes are usually stably expressed in various cells and tissues. However, it was reported that the expression of some reference genes may be distinct in different species. In this study, we intend to answer whether the expression of reported traditional reference genes changes or not in the polyploid fish RESULTS: By retrieving the mRNA sequencing data of three different ploidy fish from the NCBI SRA database, we selected 12 candidate reference genes, and examined their expression levels in the 10 tissues and in the four cell lines of three different ploidy fish by real-time PCR. Then, the expression profiles of these 12 candidate reference genes were systematically evaluated by using the software platforms: BestKeeper, NormFinder and geNorm. CONCLUSION: The 28S ribosomal protein S5 gene (RPS5) and the ribosomal protein S18 gene (RPS18) are the most suitable reference genes for the polyploid of Cyprinus carpio and Carassius auratus, demonstrated by both of the tissues and the cultured cells.
Assuntos
Carpas/genética , Carpa Dourada/genética , Poliploidia , Proteínas Ribossômicas/genética , Transcriptoma , Animais , Proteínas de Peixes/genética , Perfilação da Expressão Gênica , Reação em Cadeia da Polimerase em Tempo Real , Padrões de Referência , SoftwareRESUMO
Piroplasmosis is a serious debilitating and sometimes fatal disease. Phylogenetic relationships within piroplasmida are complex and remain unclear. In the study, we assessed the relative resolution capabilities of the DNA sequences of the nuclear genes 40S ribosomal protein S5 (RPS5) and mitochondrial DNA Cytochrome c oxidase subunit III (cox3) gene in the phylogeny of Babesia and Theileria species isolates. We demonstrated that by using the cox3 gene can recover a better supported species tree for some Theileria species than when using the nuclear RPS5 gene alone, it tends to intra-specific diversity and considerable inter-specific difference. Additionally, the combined DNA sequences of the nuclear RPS5 and cox3 gene improved the inference of evolutionary relationships among Babesia and Theileria species. The mitochondrial cox3 gene outperforms nuclear RPS5 gene and yields better resolution on the intra-specific diversity of Babesia and Theileria species. However, the combined RPS5 nuclear DNA and cox3 DNA tree had more advantage in the phylogeny of Babesia and Theileria species than that of single gene alone.
Assuntos
Babesia/classificação , Complexo IV da Cadeia de Transporte de Elétrons/genética , Filogenia , Proteínas Ribossômicas/genética , Theileria/classificação , Animais , Babesia/genética , Sequência de Bases , Biodiversidade , Bovinos , DNA Mitocondrial/fisiologia , DNA de Protozoário/fisiologia , Marcadores Genéticos , Alinhamento de Sequência , Ovinos , Organismos Livres de Patógenos Específicos , Theileria/genéticaRESUMO
Plastid ribosomal proteins are essential components of protein synthesis machinery and have diverse roles in plant growth and development. Mutations in plastid ribosomal proteins lead to a range of developmental phenotypes in plants. However, how they regulate these processes is not fully understood, and the functions of some individual plastid ribosomal proteins remain unknown. To identify genes responsible for chloroplast development, we isolated and characterized a mutant that exhibited pale yellow inner leaves with a reduced growth rate in Arabidopsis. The mutant (rps5) contained a missense mutation of plastid ribosomal protein S5 (RPS5), which caused a dramatically reduced abundance of chloroplast 16S rRNA and seriously impaired 16S rRNA processing to affect ribosome function and plastid translation. Comparative proteomic analysis revealed that the rps5 mutation suppressed the expression of a large number of core components involved in photosystems I and II as well as many plastid ribosomal proteins. Unexpectedly, a number of proteins associated with cold stress responses were greatly decreased in rps5, and overexpression of the plastid RPS5 improved plant cold stress tolerance. Our results indicate that RPS5 is an important constituent of the plastid 30S subunit and affects proteins involved in photosynthesis and cold stress responses to mediate plant growth and development.
Assuntos
Proteínas de Arabidopsis/fisiologia , Arabidopsis/fisiologia , Fotossíntese , Arabidopsis/crescimento & desenvolvimento , Proteínas de Arabidopsis/genética , Temperatura Baixa , Mutação de Sentido Incorreto , Fotossíntese/fisiologia , Plastídeos/fisiologia , RNA Ribossômico 16S/metabolismo , Reação em Cadeia da Polimerase em Tempo Real , Estresse Fisiológico/fisiologiaRESUMO
CircRNAs (circular RNAs) reduce the abundance of miRNAs through ceRNA (competing endogenous RNA), to regulate many physiological processes and stress responses in plants. However, the role of circRNA in drought stress is poorly understood. Through ring identification and sequencing verification of ath-circ032768, bioinformatics analysis predicted the interaction of ath-circ032768-miR472-RPS5, and further obtained transgenic plants overexpressing ath-circ032768 and silencing STTM-miR472. The change in drought stress was analysed using biochemical and molecular biological methods. Sequencing and biological analysis confirmed that ath-circ032768, miR472 and RPS5 were responsive to drought stress, and changes in gene expression were consistent with the prediction of ceRNA. The silencing vectors ath-circ032768 and STTM-miR472 were constructed using molecular biology techniques, and stable transgenic plants with drought tolerance obtained. Further physiological and biochemical studies showed that ath-circ032768 could bind to miR472, and that miR472 could bind to the RPS5 gene, resulting in decreased expression of RPS5. Hence, ath-circ032768 can competitively inhibit degradation of RPS5 by miR472 through ceRNA. This process is accompanied by increased expression of DREB2A, RD29A and RD29B genes. Through the ath-circ032768-miR472-RPS5 pathway, the RPS5 stress resistance protein interacts with DREB2A protein to enhance expression of downstream drought resistance genes, RD29A and RD29B, and participate in the regulation mechanism of plant drought resistance, thereby improving drought tolerance of plants.
Assuntos
Arabidopsis , Secas , Regulação da Expressão Gênica de Plantas , MicroRNAs , Plantas Geneticamente Modificadas , RNA Circular , MicroRNAs/genética , MicroRNAs/metabolismo , RNA Circular/genética , RNA Circular/metabolismo , Arabidopsis/genética , Arabidopsis/fisiologia , Plantas Geneticamente Modificadas/genética , Estresse Fisiológico/genética , RNA de Plantas/genética , RNA Endógeno CompetitivoRESUMO
Gene targeting (GT) is a promising tool for precise manipulation of genome sequences, however, GT in seed plants remains a challenging task. The simple and direct way to improve the efficiency of GT via homology-directed repair (HDR) is to increase the frequency of double-strand breaks (DSBs) at target sites in plants. Here we report an all-in-one approach of GT in Arabidopsis by combining a transcriptional and a translational enhancer for the Cas expression. We find that facilitating the expression of Cas9 and Cas12a variant by using enhancers can improve DSB and subsequent knock-in efficiency in the Arabidopsis genome. These results indicate that simply increasing Cas protein expression at specific timings - egg cells and early embryos - can improve the establishment of heritable GTs. This simple approach allows for routine genome engineering in plants.
RESUMO
BACKGROUND: Previous investigations have shown that exosome secretion from hypoxic pre-treated adipose-derived stem cells (ADSCs) affect ischemic injury treatment; however, the therapeutic effect relative to circRNA delivery is unclear. METHODS: In the present investigation inflammatory factors, nerve injury, and cognitive function were assessed using a middle cerebral artery occlusion mouse model. The isolated exosomes were identified using transmission electron microscopy and further tested by leveraging exosome particles in a nanoparticle tracking approach. Differences in circRNA expression between exosomes and hypoxic pre-treated ADSC exosomes were analyzed by high-throughput sequencing. The phenotypic transformation of microglia was detected by immunofluorescence. The circRNA and downstream target were analyzed by bioinformatics, RT-qPCR, and luciferase report. RESULTS: Exosomes from hypoxic pre-treated ADSCs improved cognitive function by reducing neuronal damage in the hippocampus after cerebral infarction. Exosomes from hypoxic pre-treated ADSCs improved cognitive function via delivery of circ-Rps5. SIRT7 and miR-124-3p were circ-Rps5 downstream targets, which was confirmed by luciferase report analysis. miR-124-3p overexpression or SIRT7 downregulation reversed the circ-Rps5-mediated M2 microglial shift under LPS conditions. Circ-Rps5-modified ADSC exosome improved cognitive function by decreasing neuronal damage and shifting microglia from an M1 to M2 phenotype in the hippocampus. CONCLUSION: The study showed that exosomes from hypoxic pre-treated ADSCs attenuated acute ischemic stroke-induced brain injury via delivery of circ-Rps5 and promoted M2 microglia/macrophage polarization.
Assuntos
Exossomos/transplante , Infarto da Artéria Cerebral Média/terapia , Macrófagos/metabolismo , Células-Tronco Mesenquimais/metabolismo , Microglia/metabolismo , RNA Circular/metabolismo , Proteínas Ribossômicas/genética , Tecido Adiposo/citologia , Animais , Células Cultivadas , Cognição , Exossomos/metabolismo , Hipocampo/citologia , Hipocampo/metabolismo , Infarto da Artéria Cerebral Média/metabolismo , Masculino , Células-Tronco Mesenquimais/efeitos dos fármacos , Camundongos , Camundongos Endogâmicos C57BL , MicroRNAs/genética , MicroRNAs/metabolismo , RNA Circular/genética , Sirtuínas/genética , Sirtuínas/metabolismoRESUMO
[This corrects the article DOI: 10.3389/fphar.2018.00022.].
RESUMO
Edwardsiella piscicida is found to be an important facultative intracellular pathogen with a broad host range. These organisms can replicate and survive within host macrophages to escape from the subversion of the immune defense. E. piscicida-macrophage interaction is very important in determining the outcome of edwardsiellasis. As an effector protein of E. piscicida T6SS, EvpP has been determined to be a very important virulence factor for E. piscicida, although its precise role in E. piscicida-macrophage interactions is not yet clear. In this study, the roles of EvpP in E. piscicida-macrophage interactions were characterized. Here, we constructed the deletion mutants of evpP (ΔevpP) and complementation (ΔevpP-C) by the allelic exchange method. Compared to wild type strain (WT), ΔevpP was found to be attenuated for growth within macrophages. In line with this observation, we found its survival capacity was lower than WT under oxidative and acid stress in vitro, which simulate conditions encountered in host macrophages. Attenuation of ΔevpP also correlated with enhanced activation of macrophages, as reflected by augmented NO production in ΔevpP-treated macrophages. Moreover, compared to WT, ΔevpP induced markedly increased apoptosis of macrophages, characterized by increased Annexin V binding and the activation of cleaved caspase-3. These findings provided strong evidence that EvpP is involved in the process of E. piscicida-macrophage interactions and is required for its survival and replication in macrophages. Thus, we propose that EvpP might be an important factor that controlling the fate of E. piscicida inside macrophages. To further exploring the underlying mechanism of EvpP action, the cDNA library was constructed from E. piscicida-infected macrophages and a yeast two-hybrid screen was performed to search for cellular proteins interacting with EvpP. Ribosomal protein S5 (RPS5) was identified as a target of EvpP. Furthermore, the interaction was validated with co-immunoprecipitation assay. This result implies that the observed effect of EvpP on macrophages might be related to RPS5-mediated regulation, contributing to a better understanding of the mechanisms of EvpP involved in E. piscicida-macrophage interactions.
Assuntos
Infecções por Enterobacteriaceae , Doenças dos Peixes , Animais , Proteínas de Bactérias/genética , Edwardsiella , MacrófagosRESUMO
Focusing on the discovery and characterization of the Arabidopsis disease resistance protein RPS5 and its guardee PBS1, this review discusses work done in the Innes laboratory from the initial identification of the RPS5 gene in 1995 to the recent deployment of the PBS1 decoy system in crops. This is done through discussion of the structure, function, and signaling environment of RPS5 and PBS1, highlighting collaborations and influential ideas along the way. RPS5, a nucleotide-binding leucine-rich repeat (NLR) protein, is activated by the proteolytic cleavage of PBS1. We have shown that the cleavage site within PBS1 can be altered to contain cleavage sites for other proteases, enabling RPS5 activation by these proteases, thereby conferring resistance to different pathogens. This decoy approach has since been translated into crop species using endogenous PBS1 orthologs and holds strong potential for GMO-free development of new genetic resistance against important crop pathogens.
Assuntos
Proteínas de Arabidopsis , Arabidopsis , Proteínas de Bactérias , Resistência à Doença , Humanos , Proteínas Serina-Treonina QuinasesRESUMO
Rabbit hemorrhagic disease virus (RHDV), a member of Caliciviridae family, causes a highly contagious disease in rabbits. The RHDV replication mechanism is poorly understood due to the lack of a suitable culture system in vitro. This study identified RHDV 5' and 3' extremities (Ex) RNA binding proteins from the rabbit kidney cell line RK-13 based on a pull-down assay by applying a tRNA scaffold streptavidin aptamer. Using mass spectrometry (MS), several host proteins were discovered which interact with RHDV 5' and 3' Ex RNA. The ribosomal protein S5 (RPS5) was shown to interact with RHDV 3' Ex RNA directly by RNA-pulldown and confocal microscopy. To further investigate the role of RPS5 in RHDV replication, small interfering RNAs for RPS5 and RPS5 eukaryotic expression plasmids were used to change the expression level of RPS5 in RK-13 cells and the results showed that the RHDV replication and translation levels were positively correlated with the expression level of RPS5. It was also verified that RPS5 promoted RHDV replication by constructing RPS5 stable overexpression cell lines and RPS5 knockdown cell lines. In summary, it has been identified that RPS5 interacted with the RHDV 3' Ex RNA region and played a role in virus replication. These results will help to understand the mechanism of RHDV replication.
Assuntos
Infecções por Caliciviridae/veterinária , Vírus da Doença Hemorrágica de Coelhos/metabolismo , Proteínas Ribossômicas/metabolismo , Replicação Viral/genética , Infecções por Caliciviridae/virologia , Regulação Viral da Expressão Gênica , Vírus da Doença Hemorrágica de Coelhos/genéticaRESUMO
Arhuaco Adams & Bernard (1977) is one of the least known genera of Neotropical Satyrinae. It comprises two species and presents an unusual disjunct distribution, with A. ica Adams & Bernard (1977), endemic to the isolated Colombian Sierra Nevada de Santa Marta, and A. dryadina (Schaus 1913) found in the mountains of Costa Rica and Panama. Here, the female of A. dryadina is described, and a new generic diagnosis is presented. Affinities with other genera of the subtribe Pronophilina, in particular the potential closest relatives, such as Pronophila Doubleday (1849), are investigated based on morphological, molecular, ecological, and behavioral data. Results from molecular and morphological sources are incongruent. Molecular data indicate that Arhuaco is paraphyletic, with A. dryadina segregating within the Pronophila clade. Morphological data, by contrast, indicate a closer affinity between the two species currently placed in Arhuaco, favoring the monophyly of the genus, and show no consistent synapomorphies for Arhuaco + Pronophila. A vicariance biogeographical scenario is evaluated.
Assuntos
Borboletas/anatomia & histologia , Borboletas/classificação , Filogenia , Animais , Colômbia , Costa Rica , DNA Mitocondrial/genética , Ecossistema , Feminino , Masculino , PanamáRESUMO
Post-menopausal osteoporosis (PMOP) is a metabolic bone disorder characterized by low bone mass and micro-architectural deterioration of bone tissue. The over-activated osteoclastogenesis, which plays an important role in osteoporosis, has become an important therapeutic target. M54 was a bioactive derivative of the Chinese traditional herb matrine. We found that M54 could suppress RANKL-induced osteoclastogenesis in bone marrow mononuclear cells and RAW264.7 cells through suppressing NF-κB, PI3K/AKT, and MAPKs pathways activity in vitro, and prevent ovariectomy-induced bone loss in vivo. Our previous study has proved that ribosomal protein S5 (RPS5) was a direct target of M19, based on which M54 was synthesized. Thus we deduced that M54 also targeted RPS5. During osteoclastogenesis, the RPS5 level in RAW264.7 cells was significantly down-regulated while M54 could maintain its level. After RPS5 was silenced, the inhibitory effects of M54 on osteoclastogenesis were partially compromised, indicating that M54 took effects through targeting RPS5. In summary, M54 was a potential clinical medicine for post-menopause osteoporosis treatment, and RPS5 is a possible key protein in PMOP.