RESUMO
Transcription factors (TFs) regulate gene programs, thereby controlling diverse cellular processes and cell states. To comprehensively understand TFs and the programs they control, we created a barcoded library of all annotated human TF splice isoforms (>3,500) and applied it to build a TF Atlas charting expression profiles of human embryonic stem cells (hESCs) overexpressing each TF at single-cell resolution. We mapped TF-induced expression profiles to reference cell types and validated candidate TFs for generation of diverse cell types, spanning all three germ layers and trophoblasts. Targeted screens with subsets of the library allowed us to create a tailored cellular disease model and integrate mRNA expression and chromatin accessibility data to identify downstream regulators. Finally, we characterized the effects of combinatorial TF overexpression by developing and validating a strategy for predicting combinations of TFs that produce target expression profiles matching reference cell types to accelerate cellular engineering efforts.
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Diferenciação Celular , Fatores de Transcrição , Humanos , Cromatina , Regulação da Expressão Gênica , Células-Tronco Embrionárias Humanas/metabolismo , Fatores de Transcrição/metabolismo , Atlas como AssuntoRESUMO
T helper type 2 (Th2) cells are important regulators of mammalian adaptive immunity and have relevance for infection, autoimmunity, and tumor immunology. Using a newly developed, genome-wide retroviral CRISPR knockout (KO) library, combined with RNA-seq, ATAC-seq, and ChIP-seq, we have dissected the regulatory circuitry governing activation and differentiation of these cells. Our experiments distinguish cell activation versus differentiation in a quantitative framework. We demonstrate that these two processes are tightly coupled and are jointly controlled by many transcription factors, metabolic genes, and cytokine/receptor pairs. There are only a small number of genes regulating differentiation without any role in activation. By combining biochemical and genetic data, we provide an atlas for Th2 differentiation, validating known regulators and identifying factors, such as Pparg and Bhlhe40, as part of the core regulatory network governing Th2 helper cell fates.
Assuntos
Receptor Cross-Talk/imunologia , Células Th2/imunologia , Células Th2/metabolismo , Animais , Sistemas CRISPR-Cas/genética , Diferenciação Celular/genética , Diferenciação Celular/imunologia , Cromatina , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas/genética , Genoma , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Ativação Linfocitária/genética , Ativação Linfocitária/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Linfócitos T Auxiliares-Indutores/metabolismo , Fatores de Transcrição/metabolismoRESUMO
Kinase and phosphatase overexpression drives tumorigenesis and drug resistance. We previously developed a mass-cytometry-based single-cell proteomics approach that enables quantitative assessment of overexpression effects on cell signaling. Here, we applied this approach in a human kinome- and phosphatome-wide study to assess how 649 individually overexpressed proteins modulated cancer-related signaling in HEK293T cells in an abundance-dependent manner. Based on these data, we expanded the functional classification of human kinases and phosphatases and showed that the overexpression effects include non-catalytic roles. We detected 208 previously unreported signaling relationships. The signaling dynamics analysis indicated that the overexpression of ERK-specific phosphatases sustains proliferative signaling. This suggests a phosphatase-driven mechanism of cancer progression. Moreover, our analysis revealed a drug-resistant mechanism through which overexpression of tyrosine kinases, including SRC, FES, YES1, and BLK, induced MEK-independent ERK activation in melanoma A375 cells. These proteins could predict drug sensitivity to BRAF-MEK concurrent inhibition in cells carrying BRAF mutations.
Assuntos
Carcinogênese/genética , Melanoma/genética , Monoéster Fosfórico Hidrolases/genética , Fosfotransferases/genética , Proteínas Proto-Oncogênicas B-raf/genética , Proliferação de Células/genética , Resistencia a Medicamentos Antineoplásicos/genética , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Células HEK293 , Humanos , Melanoma/enzimologia , Melanoma/patologia , Mutação , Fosforilação/genética , Inibidores de Proteínas Quinases/farmacologia , Proteômica , Transdução de Sinais/efeitos dos fármacosRESUMO
Cells save their energy during nitrogen starvation by selective autophagy of ribosomes and degradation of RNA to ribonucleotides and nucleosides. Nucleosides are hydrolyzed by nucleoside N-ribohydrolases (nucleosidases, NRHs). Subclass I of NRHs preferentially hydrolyzes the purine ribosides while subclass II is more active towards uridine and xanthosine. Here, we performed a crystallographic and kinetic study to shed light on nucleoside preferences among plant NRHs followed by in vivo metabolomic and phenotyping analyses to reveal the consequences of enhanced nucleoside breakdown. We report the crystal structure of Zea mays NRH2b (subclass II) and NRH3 (subclass I) in complexes with the substrate analog forodesine. Purine and pyrimidine catabolism are inseparable because nucleobase binding in the active site of ZmNRH is mediated via a water network and is thus unspecific. Dexamethasone-inducible ZmNRH overexpressor lines of Arabidopsis thaliana, as well as double nrh knockout lines of moss Physcomitrium patents, reveal a fine control of adenosine in contrast to other ribosides. ZmNRH overexpressor lines display an accelerated early vegetative phase including faster root and rosette growth upon nitrogen starvation or osmotic stress. Moreover, the lines enter the bolting and flowering phase much earlier. We observe changes in the pathways related to nitrogen-containing compounds such as ß-alanine and several polyamines, which allow plants to reprogram their metabolism to escape stress. Taken together, crop plant breeding targeting enhanced NRH-mediated nitrogen recycling could therefore be a strategy to enhance plant growth tolerance and productivity under adverse growth conditions.
Assuntos
Arabidopsis , Nucleosídeos , Nucleosídeos/metabolismo , Nitrogênio/metabolismo , Melhoramento Vegetal , Plantas/metabolismo , Uridina/metabolismo , Arabidopsis/genéticaRESUMO
Mitogen-activated protein kinase kinases (MAPKKs) play a critical role in the mitogen-activated protein kinase (MAPK) signaling pathway, transducing external stimuli into intracellular responses and enabling plant adaptation to environmental challenges. Most research has focused on the model plant Arabidopsis (Arabidopsis thaliana). The systematic analysis and characterization of MAPKK genes across different plant species, particularly in cotton (Gossypium hirsutum), are somewhat limited. Here, we identified MAPKK family members from 66 different species, which clustered into 5 different sub-groups, and MAPKKs from four cotton species clustered together. Through further bioinformatic and expression analysis, GhMAPKK5 was identified as the most responsive MAPKK member to salt and drought stress among the 23 MAPKKs identified in Gossypium hirsutum. Silencing GhMAPKK5 in cotton through virus-induced gene silencing (VIGS) led to quicker wilting under salt and drought conditions, while overexpressing GhMAPKK5 in Arabidopsis enhanced root growth and seed germination under these stresses, demonstrating GhMAPKK5's positive role in stress tolerance. Transcriptomics and Yeast-Two-Hybrid assays revealed a MAPK cascade signal module comprising GhMEKK (Mitogen-activated protein kinase kinase kinases)3/8/31-GhMAPKK5-GhMAPK11/23. This signaling cascade may play a role in managing drought and salt stress by regulating transcription factor genes, such as WRKYs, which are involved in the biosynthesis and transport pathways of ABA, proline, and RALF. This study is highly important for further understanding the regulatory mechanism of MAPKK in cotton, contributing to its stress tolerance and offering potential in targets for genetic enhancement.
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Sepsis-associated encephalopathy (SAE) is a frequent complication of severe systemic infection resulting in delirium, premature death, and long-term cognitive impairment. We closely mimicked SAE in a murine peritoneal contamination and infection (PCI) model. We found long-lasting synaptic pathology in the hippocampus including defective long-term synaptic plasticity, reduction of mature neuronal dendritic spines, and severely affected excitatory neurotransmission. Genes related to synaptic signaling, including the gene for activity-regulated cytoskeleton-associated protein (Arc/Arg3.1) and members of the transcription-regulatory EGR gene family, were downregulated. At the protein level, ARC expression and mitogen-activated protein kinase signaling in the brain were affected. For targeted rescue we used adeno-associated virus-mediated overexpression of ARC in the hippocampus in vivo. This recovered defective synaptic plasticity and improved memory dysfunction. Using the enriched environment paradigm as a non-invasive rescue intervention, we found improvement of defective long-term potentiation, memory, and anxiety. The beneficial effects of an enriched environment were accompanied by an increase in brain-derived neurotrophic factor (BDNF) and ARC expression in the hippocampus, suggesting that activation of the BDNF-TrkB pathway leads to restoration of the PCI-induced reduction of ARC. Collectively, our findings identify synaptic pathomechanisms underlying SAE and provide a conceptual approach to target SAE-induced synaptic dysfunction with potential therapeutic applications to patients with SAE.
Assuntos
Fator Neurotrófico Derivado do Encéfalo , Disfunção Cognitiva , Proteínas do Citoesqueleto , Modelos Animais de Doenças , Hipocampo , Plasticidade Neuronal , Encefalopatia Associada a Sepse , Animais , Camundongos , Disfunção Cognitiva/etiologia , Disfunção Cognitiva/metabolismo , Disfunção Cognitiva/terapia , Disfunção Cognitiva/genética , Encefalopatia Associada a Sepse/metabolismo , Encefalopatia Associada a Sepse/etiologia , Encefalopatia Associada a Sepse/terapia , Encefalopatia Associada a Sepse/genética , Hipocampo/metabolismo , Fator Neurotrófico Derivado do Encéfalo/metabolismo , Fator Neurotrófico Derivado do Encéfalo/genética , Proteínas do Citoesqueleto/genética , Proteínas do Citoesqueleto/metabolismo , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Dependovirus/genética , Masculino , Potenciação de Longa Duração , Receptor trkB/metabolismo , Receptor trkB/genética , Vetores Genéticos/administração & dosagem , Vetores Genéticos/genética , Sinapses/metabolismoRESUMO
Engrailed-1 (EN1) is a developmental gene that encodes En1, a highly conserved transcription factor involved in regionalization during early embryogenesis and in the later maintenance of normal neurons. After birth, EN1 still plays a role in the development and physiology of the body; for example, it exerts a protective effect on midbrain dopaminergic (mDA) neurons, and loss of EN1 causes mDA neurons in the ventral midbrain to gradually die approximately 6 weeks after birth, resulting in motor and nonmotor symptoms similar to those observed in Parkinson's disease. Notably, EN1 has been identified as a possible susceptibility gene for idiopathic Parkinson's disease in humans. EN1 is involved in the processes of wound-healing scar production and tissue and organ fibrosis. Additionally, EN1 can lead to tumorigenesis and thus provides a target for the treatment of some tumors. In this review, we summarize the effects of EN1 on embryonic organ development, describe the consequences of the deletion or overexpression of the EN1 gene, and discuss the pathways in which EN1 is involved. We hope to clarify the role of EN1 as a developmental gene and present potential therapeutic targets for diseases involving the EN1 gene.
Assuntos
Proteínas de Homeodomínio , Doença de Parkinson , Humanos , Proteínas de Homeodomínio/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Neurônios/metabolismo , Regulação da Expressão Gênica , Genes Homeobox , Doença de Parkinson/genética , Doença de Parkinson/metabolismo , Doença de Parkinson/patologiaRESUMO
The termination factor Rho, an ATP-dependent RNA translocase, preempts pervasive transcription processes, thereby rendering genome integrity in bacteria. Here, we show that the loss of Rho function raised the intracellular pH to >8.0 in Escherichia coli. The loss of Rho function upregulates tryptophanase-A (TnaA), an enzyme that catabolizes tryptophan to produce indole, pyruvate, and ammonia. We demonstrate that the enhanced TnaA function had produced the conjugate base ammonia, raising the cellular pH in the Rho-dependent termination defective strains. On the other hand, the constitutively overexpressed Rho lowered the cellular pH to about 6.2, independent of cellular ammonia levels. Since Rho overexpression may increase termination activities, the decrease in cellular pH could result from an excess H+ ion production during ATP hydrolysis by overproduced Rho. Furthermore, we performed in vivo termination assays to show that the efficiency of Rho-dependent termination was increased at both acidic and basic pH ranges. Given that the Rho level remained unchanged, the alkaline pH increases the termination efficiency by stimulating Rho's catalytic activity. We conducted the Rho-mediated RNA release assay from a stalled elongation complex to show an efficient RNA release at alkaline pH, compared to the neutral or acidic pH, that supports our in vivo observation. Whereas acidic pH appeared to increase the termination function by elevating the cellular level of Rho. This study is the first to link Rho function to the cellular pH homeostasis in bacteria. IMPORTANCE The current study shows that the loss or gain of Rho-dependent termination alkalizes or acidifies the cytoplasm, respectively. In the case of loss of Rho function, the tryptophanase-A enzyme is upregulated, and degrades tryptophan, producing ammonia to alkalize cytoplasm. We hypothesize that Rho overproduction by deleting its autoregulatory DNA portion increases termination function, causing excessive ATP hydrolysis to produce H+ ions and cytoplasmic acidification. Therefore, this study is the first to unravel a relationship between Rho function and intrinsic cellular pH homeostasis. Furthermore, the Rho level increases in the absence of autoregulation, causing cytoplasmic acidification. As intracellular pH plays a critical role in enzyme function, such a connection between Rho function and alkalization will have far-reaching implications for bacterial physiology.
Assuntos
Transcrição Gênica , Triptofano , Triptofano/genética , Triptofano/metabolismo , Triptofanase/genética , Triptofanase/metabolismo , Amônia/metabolismo , Fator Rho/genética , Fator Rho/metabolismo , Escherichia coli/metabolismo , RNA/metabolismo , Homeostase , Trifosfato de Adenosina/metabolismo , Concentração de Íons de HidrogênioRESUMO
BACKGROUND: Lipoxygenase (LOX) is a multifunctional enzyme that is primarily related to plant organ growth and development, biotic and abiotic stress responses, and production of flavor-associated metabolites. In higher plants, the LOX family encompasses several isozymes with varying expression patterns between tissues and developmental stages. These affect processes including seed germination, seed storage, seedling growth, fruit ripening, and leaf senescence. LOX family genes have multiple functions in response to hormones such as methyl jasmonate (MeJA) and salicylic acid. RESULTS: In this study, we identified 30 and 95 LOX homologs in Medicago truncatula and Medicago sativa, respectively. These genes were characterized with analyses of their basic physical and chemical properties, structures, chromosomal distributions, and phylogenetic relationships to understand structural variations and their physical locations. Phylogenetic analysis was conducted for members of the three LOX subfamilies (9-LOX, type I 13-LOX, and type II 13-LOX) in Arabidopsis thaliana, Glycine max, M. truncatula, and M. sativa. Analysis of predicted promoter elements revealed several relevant cis-acting elements in MtLOX and MsLOX genes, including abscisic acid (ABA) response elements (ABREs), MeJA response elements (CGTCA-motifs), and antioxidant response elements (AREs). Cis-element data combined with transcriptomic data demonstrated that LOX gene family members in these species were most likely related to abiotic stress responses, hormone responses, and plant development. Gene expression patterns were confirmed via quantitative reverse transcription PCR. Several MtLOX genes (namely MtLOX15, MtLOX16, MtLOX20, and MtLOX24) belonging to the type I 13-LOX subfamily and other LOX genes (MtLOX7, MtLOX11, MsLOX23, MsLOX87, MsLOX90, and MsLOX94) showed significantly different expression levels in the flower tissue, suggesting roles in reproductive growth. Type I 13-LOXs (MtLOX16, MtLOX20, MtLOX21, MtLOX24, MsLOX57, MsLOX84, MsLOX85, and MsLOX94) and type II 13-LOXs (MtLOX5, MtLOX6, MtLOX9, MtLOX10, MsLOX18, MsLOX23, and MsLOX30) were MeJA-inducible and were predicted to function in the jasmonic acid signaling pathway. Furthermore, exogenous MtLOX24 expression in Arabidopsis verified that MtLOX24 was involved in MeJA responses, which may be related to insect-induced abiotic stress. CONCLUSIONS: We identified six and four LOX genes specifically expressed in the flowers of M. truncatula and M. sativa, respectively. Eight and seven LOX genes were induced by MeJA in M. truncatula and M. sativa, and the LOX genes identified were mainly distributed in the type I and type II 13-LOX subfamilies. MtLOX24 was up-regulated at 8 h after MeJA induction, and exogenous expression in Arabidopsis demonstrated that MtLOX24 promoted resistance to MeJA-induced stress. This study provides valuable new information regarding the evolutionary history and functions of LOX genes in the genus Medicago.
Assuntos
Acetatos , Arabidopsis , Ciclopentanos , Medicago truncatula , Oxilipinas , Medicago truncatula/genética , Medicago truncatula/metabolismo , Medicago sativa/genética , Estudo de Associação Genômica Ampla , Filogenia , Arabidopsis/genética , Hormônios/metabolismo , Regulação da Expressão Gênica de Plantas , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Estresse Fisiológico/genéticaRESUMO
BACKGROUND: As a key regulatory enzyme in the glycolysis pathway, pyruvate kinase (PK) plays crucial roles in multiple physiological processes during plant growth and is also involved in the abiotic stress response. However, little information is known about PKs in soybean. RESULTS: In this study, we identified 27 PK family genes against the genome of soybean cultivar Zhonghuang13. They were classified into 2 subfamilies including PKc and PKp. 22 segmental duplicated gene pairs and 1 tandem duplicated gene pair were identified and all of them experienced a strong purifying selective pressure during evolution. Furthermore, the abiotic stresses (especially salt stress) and hormone responsive cis-elements were present in the promoters of GmPK genes, suggesting their potential roles in abiotic stress tolerance. By performing the qRT-PCR, 6 GmPK genes that continuously respond to both NaCl and ABA were identified. Subsequently, GmPK21, which represented the most significant change under NaCl treatment was chosen for further study. Its encoded protein GmPK21 was localized in the cytoplasm and plasma membrane. The transgenic Arabidopsis overexpressing GmPK21 exhibited weakened salinity tolerance. CONCLUSIONS: This study provides genomic information of soybean PK genes and a molecular basis for mining salt tolerance function of PKs in the future.
Assuntos
Arabidopsis , Piruvato Quinase , Glycine max/genética , Cloreto de Sódio , Genes Duplicados , Arabidopsis/genéticaRESUMO
Salt stress is one of the major factors limiting plant growth and productivity. Many studies have shown that serine hydroxymethyltransferase (SHMT) gene play an important role in growth, development and stress response in plants. However, to date, there have been few studies on whether SHMT3 can enhance salt tolerance in plants. Therefore, the effects of overexpression or silencing of CsSHMT3 gene on cucumber seedling growth under salt stress were investigated in this study. The results showed that overexpression of CsSHMT3 gene in cucumber seedlings resulted in a significant increase in chlorophyll content, photosynthetic rate and proline (Pro) content, and antioxidant enzyme activity under salt stress condition; whereas the content of malondialdehyde (MDA), superoxide anion (H2O2), hydrogen peroxide (O2·-) and relative conductivity were significantly decreased when CsSHMT3 gene was overexpressed. However, the content of chlorophyll and Pro, photosynthetic rate, and antioxidant enzyme activity of the silenced CsSHMT3 gene lines under salt stress were significantly reduced, while MDA, H2O2, O2·- content and relative conductivity showed higher level in the silenced CsSHMT3 gene lines. It was further found that the expression of stress-related genes SOD, CAT, SOS1, SOS2, NHX, and HKT was significantly up-regulated by overexpressing CsSHMT3 gene in cucumber seedlings; while stress-related gene expression showed significant decrease in silenced CsSHMT3 gene seedlings under salt stress. This suggests that overexpression of CsSHMT3 gene increased the salt tolerance of cucumber seedlings, while silencing of CsSHMT3 gene decreased the salt tolerance. In conclusion, CsSHMT3 gene might positively regulate salt stress tolerance in cucumber and be involved in regulating antioxidant activity, osmotic adjustment, and photosynthesis under salt stress. KEY MESSAGE: CsSHMT3 gene may positively regulate the expression of osmotic system, photosynthesis, antioxidant system and stress-related genes in cucumber.
Assuntos
Clorofila , Cucumis sativus , Regulação da Expressão Gênica de Plantas , Fotossíntese , Estresse Salino , Tolerância ao Sal , Plântula , Cucumis sativus/genética , Cucumis sativus/crescimento & desenvolvimento , Cucumis sativus/fisiologia , Cucumis sativus/efeitos dos fármacos , Plântula/genética , Plântula/crescimento & desenvolvimento , Plântula/efeitos dos fármacos , Plântula/fisiologia , Regulação da Expressão Gênica de Plantas/efeitos dos fármacos , Tolerância ao Sal/genética , Estresse Salino/genética , Clorofila/metabolismo , Fotossíntese/genética , Fotossíntese/efeitos dos fármacos , Peróxido de Hidrogênio/metabolismo , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Glicina Hidroximetiltransferase/genética , Glicina Hidroximetiltransferase/metabolismo , Antioxidantes/metabolismo , Malondialdeído/metabolismo , Plantas Geneticamente Modificadas , Inativação GênicaRESUMO
We present an economic and straightforward method to introduce 13C-19F spin systems into the deuterated aromatic side chains of phenylalanine as reporters for various protein NMR applications. The method is based on the synthesis of [4-13C, 2,3,5,6-2H4] 4-fluorophenylalanine from the commercially available isotope sources [2-13C] acetone and deuterium oxide. This compound is readily metabolized by standard Escherichia coli overexpression in a glyphosate-containing minimal medium, which results in high incorporation rates in the corresponding target proteins.
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The normal development of pollen grains and the completion of double fertilization in embryos are crucial for both the sexual reproduction of angiosperms and grain production. Actin depolymerizing factor (ADF) regulates growth, development, and responses to biotic and abiotic stress by binding to actin in plants. In this study, the function of the ZmADF1 gene was validated through bioinformatic analysis, subcellular localization, overexpression in maize and Arabidopsis, and knockout via CRISPR/Cas9. The amino acid sequence of ZmADF1 exhibited high conservation and a similar tertiary structure to that of ADF homologs. Subcellular localization analysis revealed that ZmADF1 is localized mainly to the nucleus and cytoplasm. The ZmADF1 gene was specifically expressed in maize pollen, and overexpression of the ZmADF1 gene decreased the number of pollen grains in the anthers of transgenic Arabidopsis plants. The germination rate of pollen and the empty seed shell rate in the fruit pods of the overexpressing plants were significantly greater than those in the wild-type (WT) plants. In maize, the pollen viability of the knockout lines was significantly greater than that of both the WT and the overexpressing lines. Our results confirmed that the ZmADF1 gene was specifically expressed in pollen and negatively regulated pollen quantity, vigor, germination rate, and seed setting rate. This study provides insights into ADF gene function and possible pathways for improving high-yield maize breeding.
Assuntos
Arabidopsis , Destrina , Pólen , Zea mays , Sequência de Aminoácidos , Arabidopsis/metabolismo , Destrina/genética , Destrina/metabolismo , Gelsolina/metabolismo , Regulação da Expressão Gênica de Plantas , Pólen/genética , Pólen/crescimento & desenvolvimento , Zea mays/metabolismoRESUMO
BACKGROUND: Soil salinity is one of the primary environmental stresses faced in rice production. When plants are exposed to salt stress, a series of cellular balances will be disrupted. Dufulin is an immune-induced antiviral agent used in plants. The DUF gene family influences plant response to abiotic stress, and the functional role of OsDUF6(ABA98726.1) in rice response to salt stress is being investigated here. RESULTS: Based on the transcriptome analysis of Dufulin treatment in inducing salt tolerance in rice, we selected the OsDUF6 protein located on the cell membrane and studied its molecular function by overexpressing OsDUF6. Salt-induced decreases in root, stem, and leaf length and increased leaf yellowing rate and Na+ concentration in the wild-type plant were mitigated in the overexpressed lines. OsDUF6 overexpression increased the enzymatic antioxidant activities of superoxide dismutase, peroxidase, catalase, and phenylalanine ammonia-lyase. OsDUF6 also played a positive role in Na+ transport as reflected by the increased growth of a salt-sensitive yeast mutant complemented with OsDUF6 in the presence of salt stress. In addition, Reverse transcription quantitative PCR analysis confirmed that the overexpression of OsDUF6 significantly changed the expression level of other genes related to growth and stress tolerance. CONCLUSIONS: Combined with previously published data, our results supported the observation that OsDUF6 is an important functional factor in Dufulin-induced promotion of salt stress tolerance in rice.
Assuntos
Oryza , Tolerância ao Sal , Tolerância ao Sal/genética , Oryza/genética , Plantas Geneticamente Modificadas/genética , Proteínas de Plantas/genética , Estresse Salino , Estresse Fisiológico/genética , Regulação da Expressão Gênica de PlantasRESUMO
MAIN CONCLUSION: Excess of KRP4 in the developing kernels in rice causes poor filling of the grains possibly through inhibition of CDKA;2 and CDKB;1 activity mediated by its interaction with CDKF;3. The potential yield of the rice varieties producing compact and heavy panicles bearing numerous spikelets is compromised because a high percentage of spikelets remain poorly filled, reportedly because of a high expression of KRPs that causes suppression of endosperm cell proliferation. To test the stated negative relationship between KRP expression and grain filling, Orysa;KRP4 was overexpressed under the control of seed-specific glutelin promoter in IR-64 rice variety that shows good grain filling. The transgenic lines showed more than 15-fold increase in expression of KRP4 in the spikelets concomitant with nearly 50% reduction in grain filling compared with the wild type without producing any significant changes on the other yield-related parameters like panicle length and the spikelets numbers that were respectively 30.23 ± 0.89 cm and 229.25 ± 33.72 per panicle in the wild type, suggesting a highly organ-targeted effect of the genetic transformation. Yeast two-hybrid test revealed CDKF;3 as the interacting partner of KRP4, and CDKF;3 was found to interact with CDKA;2, CDKB;1 and CDKD;1. Significant decrease in grain filling in the transgenic lines compared with the wild type due to overexpression of KRP4 could be because of suppression of the activity of CDKB;1 and CDKA;2 by inhibition of their phosphorylation directly by CDKF;3, or mediated through inhibition of phosphorylation of CDKD;1 by CDKF;3. The study thus indicated that suppression of expression of KRP(s) by genetic manipulation of their promoters could be an important way of improving the yield of the rice varieties bearing compact and heavy panicles.
Assuntos
Grão Comestível , Regulação da Expressão Gênica de Plantas , Oryza , Proteínas de Plantas , Plantas Geneticamente Modificadas , Sementes , Oryza/genética , Oryza/metabolismo , Oryza/crescimento & desenvolvimento , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Sementes/genética , Sementes/crescimento & desenvolvimento , Sementes/metabolismo , Grão Comestível/genética , Grão Comestível/crescimento & desenvolvimento , Grão Comestível/metabolismo , Regiões Promotoras Genéticas/genética , Técnicas do Sistema de Duplo-Híbrido , Quinases Ciclina-Dependentes/metabolismo , Quinases Ciclina-Dependentes/genéticaRESUMO
Paclitaxel is one of the most effective anticancer drugs ever developed. Although the most sustainable approach to its production is provided by plant cell cultures, the yield is limited by bottleneck enzymes in the taxane biosynthetic pathway: baccatin-aminophenylpropanoyl-13-O-transferase (BAPT) and 3'-N-debenzoyltaxol N-benzoyltransferase (DBTNBT). With the aim of enhancing paclitaxel production by overcoming this bottleneck, we obtained distinct lines of Taxus baccata in vitro roots, each independently overexpressing either of the two flux-limiting genes, BAPT or DBTNBT, through a Rhizobium rhizogenes A4-mediated transformation. Due to the slow growth rate of the transgenic Taxus roots, they were dedifferentiated to obtain callus lines and establish cell suspensions. The transgenic cells were cultured in a two-stage system and stimulated for taxane production by a dual elicitation treatment with 1 µm coronatine plus 50 mm of randomly methylated-ß-cyclodextrins. A high overexpression of BAPT (59.72-fold higher at 48 h) and DBTNBT (61.93-fold higher at 72 h) genes was observed in the transgenic cell cultures, as well as an improved taxane production. Compared to the wild type line (71.01 mg/L), the DBTNBT line produced more than four times higher amounts of paclitaxel (310 mg/L), while the content of this taxane was almost doubled in the BAPT line (135 mg/L). A transcriptional profiling of taxane biosynthetic genes revealed that GGPPS, TXS and DBAT genes were the most reactive to DBTNBT overexpression and the dual elicitation, their expression increasing gradually and constantly. The same genes exhibited a pattern of isolated peaks of expression in the elicited BAPT-overexpressing line.
Assuntos
Paclitaxel , Taxus , Paclitaxel/metabolismo , Taxus/genética , Taxus/metabolismo , Células Cultivadas , Taxoides/farmacologia , Taxoides/metabolismoRESUMO
Streptocarbazoles are a class of indolocarbazole (ICZ) compounds produced by Streptomyces strains that feature unique cyclic N-glycosidic linkages between the 1,3-carbon atoms of the glycosyl moiety and the two indole nitrogen atoms. Although several streptocarbazole compounds display effective cytotoxic activity, their biosynthesis remains unclear. Herein, through the inactivation of the aminotransferase gene spcI in the staurosporine biosynthetic gene cluster spc followed by heterologous expression, two new streptocarbazole derivatives (1 and 3) and three known ICZs (2, 4, and 5) were generated. Their structures were determined by a combination of spectroscopic methods, circular dichroism measurements, and single-crystal X-ray diï¬raction. Compounds 1-4 displayed moderate cytotoxicity against HCT-116 cell line, and compounds 3 and 4 were effective against Huh 7 cell line. Double-gene knockout experiments allowed us to propose a biosynthetic pathway for streptocarbazole productions. Furthermore, by overexpression of the involving key enzymes, the production of streptocarbazoles 1 and 3 were improved by approximately 1.5-2.5 fold. IMPORTANCE: Indolocarbazoles (ICZs) are a group of antitumor agents, with several analogs used in clinical trials. Therefore, the identification of novel ICZ compounds is important for drug discovery. Streptocarbazoles harbor unique N-glycosidic linkages (N13-C1' and N12-C3'), distinguishing them from the representative ICZ compound staurosporine; however, their biosynthesis remains unclear. In this study, two new streptocarbazoles (1 and 3) with cytotoxic activities were obtained by manipulating the staurosporine biosynthetic gene cluster spc followed by heterologous expression. The biosynthetic pathway of streptocarbazoles was proposed, and their productions were improved through the overexpression of the key enzymes involved. This study enriches the structural diversity of ICZ compounds and would facilitate the discovery of new streptocarbazoles via synthetic biological strategies.
Assuntos
Carbazóis , Streptomyces , Estaurosporina/farmacologia , Carbazóis/farmacologia , Carbazóis/química , Carbazóis/metabolismo , Streptomyces/metabolismo , Família MultigênicaRESUMO
INTRODUCTION: Exploring metabolic changes within host E. coli through an untargeted metabolomic study of T7L variants overexpression to optimize engineered endolysins for clinical/therapeutic use. AIM AND OBJECTIVE: This study aims to assess the impact of overexpressing T7L variants on the metabolic profiles of E. coli. The two variants considered include T7L-H37A, which has enhanced lytic activity compared to its wild-type protein, and T7L-H48K, a dead mutant with no significant activity. METHODS: 1H NMR-based metabolomics was employed to compare the metabolic profiles of E. coli cells overexpressing T7L wild-type protein and its variants. RESULTS: Overexpression of the T7L wild-type (T7L-WT) protein and its variants (T7L-H48K and T7L-H37A) was compared to RNAP overexpression in E. coli cells using 1H NMR-based metabolomics, analyzing a total of 75 annotated metabolites, including organic acids, amino acids, sugars, and nucleic acids. The results showed distinct clustering patterns for the two T7L variant groups compared with the WT, in which the dead mutant (H48K) group showed clustering close to that of RNAP. Pathway impact analysis revealed different effects of T7L variants on E. coli metabolic profiles, with T7L-H48K showing minimal alterations in energy and amino acid pathways linked to osmotic stress compared to noticeable alterations in these pathways for both T7L-H37A and T7L-WT. CONCLUSIONS: This study uncovered distinct metabolic fingerprints when comparing the overexpression of active and inactive mutants of T7L lytic enzymes in E. coli cells. These findings could contribute to the optimization and enhancement of suitable endolysins as potential alternatives to antibiotics.
Assuntos
Escherichia coli , Metaboloma , Metabolômica , Escherichia coli/metabolismo , Escherichia coli/genética , Metabolômica/métodos , Proteínas Virais/metabolismo , Proteínas Virais/genética , Bacteriófago T7/genética , Bacteriófago T7/metabolismo , Mutação , RNA Polimerases Dirigidas por DNA/metabolismo , RNA Polimerases Dirigidas por DNA/genéticaRESUMO
Microalgae and cyanobacteria are a rich source of carotenoids that are well known for their potent bioactivities, including antioxidant, anti-cancer, anti-proliferative, anti-inflammatory, and anti-obesity properties. Recently, many interests have also been focused on the biological activities of these microalgae/cyanobacteria-derived carotenoids, such as fucoxanthin and ß-carotene potential to be the salutary nutraceuticals, on treating or preventing human common diseases (e.g., cancers). This is due to their special chemical structures that demonstrate unique bioactive functions, in which the biologically active discrepancies might attribute to the different spatial configurations of their molecules. In addition, their abundance and bioaccessibilities make them more popularly applied in food and pharmaceutical industries, as compared to the macroalgal/fungal-derived ones. This review is focused on the recent studies on the bioactivities of fucoxanthin and some carotenoids derived from microalgae and cyanobacteria in relationship with human health and diseases, with emphasis on their potential applications as natural antioxidants. Various biotechnological approaches employed to induce the production of these specific carotenoids from the culture of microalgae/cyanobacteria are also critically reviewed. These well-developed and emerging biotechnologies present promise to be applied in food and pharmaceutical industries to facilitate the efficient manufacture of the bioactive carotenoid products derived from microalgae and cyanobacteria.
RESUMO
Thioredoxin-1 (Trx1) has cardioprotective effects on ischemia/reperfusion (I/R) injury, although its role in ischemic postconditioning (PostC) in middle-aged mice is not understood. This study aimed to evaluate if combining two cardioprotective strategies, such as Trx1 overexpression and PostC, could exert a synergistic effect in reducing infarct size in middle-aged mice. Young or middle-aged wild-type mice (Wt), transgenic mice overexpressing Trx1, and dominant negative (DN-Trx1) mutant of Trx1 mice were used. Mice hearts were subjected to I/R or PostC protocol. Infarct size, hydrogen peroxide (H2O2) production, protein nitration, Trx1 activity, mitochondrial function, and Trx1, pAkt and pGSK3ß expression were measured. PostC could not reduce infarct size even in the presence of Trx1 overexpression in middle-aged mice. This finding was accompanied by a lack of Akt and GSK3ß phosphorylation, and Trx1 expression (in Wt group). Trx1 activity was diminished and H2O2 production and protein nitration were increased in middle-age. The respiratory control rate dropped after I/R in Wt-Young and PostC restored this value, but not in middle-aged groups. Our results showed that Trx1 plays a key role in the PostC protection mechanism in young but not middle-aged mice, even in the presence of Trx1 overexpression.