RESUMO
As an information bridge between DNA and protein, RNA regulates cellular processes and gene expression in various ways. From its synthesis to degradation, RNA is associated with a range of RNA-binding proteins. Therefore, it is necessary to develop innovative methods to study the interaction between RNA and proteins. Previously, we developed an RNA-centric method, called CRISPR-based RNA-United Interacting System (CRUIS), to capture RNA-protein interaction in cells. On this basis, here we develop an enhanced CRUIS (eCRUIS) by combining the power of dCas13d and the engineered promiscuous ligase TurboID. The current version allows us to rapidly label RNA-binding proteins on the target RNA within 30 minutes, potentially for in vivo use. By introducing bait-assay with exogenous RNA, we confirm that eCRUIS can effectively label RNA-binding proteins on bait RNA in a short time. eCRUIS provides a broader range of in vitro and in vivo applications for studying RNA-protein interactions.
Assuntos
Sistemas CRISPR-Cas , Proteínas de Ligação a RNA , Humanos , Sistemas CRISPR-Cas/genética , Células HEK293 , Ligação Proteica , RNA/metabolismo , RNA/genética , Proteínas de Ligação a RNA/metabolismo , Proteínas de Ligação a RNA/genéticaRESUMO
The cytoophidium is an evolutionarily conserved subcellular structure formed by filamentous polymers of metabolic enzymes. In vertebrates, inosine monophosphate dehydrogenase (IMPDH), which catalyses the rate-limiting step in guanosine triphosphate (GTP) biosynthesis, is one of the best-known cytoophidium-forming enzymes. Formation of the cytoophidium has been proposed to alleviate the inhibition of IMPDH, thereby facilitating GTP production to support the rapid proliferation of certain cell types such as lymphocytes, cancer cells and pluripotent stem cells (PSCs). However, past studies lacked appropriate models to elucidate the significance of IMPDH cytoophidium under normal physiological conditions. In this study, we demonstrate that the presence of IMPDH cytoophidium in mouse PSCs correlates with their metabolic status rather than pluripotency. By introducing IMPDH2 Y12C point mutation through genome editing, we established mouse embryonic stem cell (ESC) lines incapable of forming IMPDH polymers and the cytoophidium. Our data indicate an important role of IMPDH cytoophidium in sustaining a positive feedback loop that couples nucleotide biosynthesis with upstream metabolic pathways. Additionally, we find that IMPDH2 Y12C mutation leads to decreased cell proliferation and increased DNA damage in teratomas, as well as impaired embryo development following blastocoel injection. Further analysis shows that IMPDH cytoophidium assembly in mouse embryonic development begins after implantation and gradually increases throughout fetal development. These findings provide insights into the regulation of IMPDH polymerisation in embryogenesis and its significance in coordinating cell metabolism and development.
Assuntos
Proliferação de Células , IMP Desidrogenase , Animais , Feminino , Camundongos , Dano ao DNA , Desenvolvimento Fetal/genética , Guanosina Trifosfato/metabolismo , IMP Desidrogenase/metabolismo , IMP Desidrogenase/genética , Camundongos Endogâmicos C57BL , Células-Tronco Pluripotentes/metabolismo , Células-Tronco Pluripotentes/citologia , Estruturas Celulares/metabolismoRESUMO
CTP synthase (CTPS) catalyzes the final step of de novo synthesis of CTP. CTPS was first discovered to form filamentous structures termed cytoophidia in Drosophila ovarian cells. Subsequent studies have shown that cytoophidia are widely present in cells of three life domains. In the Drosophila ovary model, our previous studies mainly focused on the early and middle stages, with less involvement in the later stages. In this work, we focus on the later stages of female germline cells in Drosophila. We use live-cell imaging to capture the continuous dynamics of cytoophidia in Stages 10-12. We notice the heterogeneity of cytoophidia in the two types of germline cells (nurse cells and oocytes), manifested in significant differences in morphology, distribution, and dynamics. Surprisingly, we also find that neighboring nurse cells in the same egg chamber exhibit multiple dynamic patterns of cytoophidia over time. Although the described dynamics may be influenced by the in vitro incubation conditions, our observation provides an initial understanding of the dynamics of cytoophidia during late-stage Drosophila oogenesis.
Assuntos
Carbono-Nitrogênio Ligases , Drosophila , Animais , Feminino , Oogênese , Citoesqueleto , OócitosRESUMO
CTP synthase (CTPS), the rate-limiting enzyme in the de novo synthesis of CTP, assembles into a filamentous structure termed the cytoophidium. The Hippo pathway regulates cell proliferation and apoptosis. The relationship of the nucleotide metabolism with the Hippo pathway is little known. Here, we study the impact of the Hippo pathway on the cytoophidium in Drosophila melanogaster posterior follicle cells (PFCs). We find that the inactivation of the Hippo pathway correlates with reduced cytoophidium length and number within PFCs. During the overexpression of CTPS, the presence of Hippo mutations also reduces the length of cytoophidia in PFCs. In addition, we observe that knocking down CTPS mitigates hpo (Hippo)-associated over-proliferation. In summary, our results suggest that there is a connection between the Hippo pathway and the nucleotide biosynthesis enzyme CTPS in PFCs.
Assuntos
Proteínas de Drosophila , Drosophila , Animais , Drosophila/metabolismo , Drosophila melanogaster/metabolismo , Via de Sinalização Hippo , Citoesqueleto/metabolismo , Nucleotídeos/metabolismo , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismoRESUMO
Cytidine triphosphate synthase (CTPS) forms cytoophidia in all three domains of life. Here we focus on the function of cytoophidia in cell proliferation using Schizosaccharomyces pombe as a model system. We find that converting His359 of CTPS into Ala359 leads to cytoophidium disassembly. By reducing the level of CTPS protein or specific mutation, the loss of cytoophidia prolongs the G2 phase and expands cell size. In addition, the loss-filament mutant of CTPS leads to a decrease in the expression of genes related to G2/M transition and cell growth, including histone chaperone slm9. The overexpression of slm9 alleviates the G2 phase elongation and cell size enlargement induced by CTPS loss-filament mutants. Overall, our results connect cytoophidia with cell cycle and cell size control in Schizosaccharomyces pombe.
Assuntos
Schizosaccharomyces , Schizosaccharomyces/genética , Ciclo Celular/genética , Divisão Celular , Proliferação de Células , Fase G2RESUMO
The conservation of agricultural heritage systems (AHSs) has played a pivotal role in fostering the sustainable development of agriculture and safeguarding farmers' livelihoods and food security worldwide. This significance is particularly evident in the case of tea AHSs, due to the economic and nutritional value of tea products. Taking the Anxi Tieguanyin Tea Culture System (ATTCS) and Fuding White Tea Culture System (FWTCS) in Fujian Province as examples, this study uses statistical analyses and a multinomial logistic regression model to assess and compare farmer livelihood and food security at the tea AHS sites. The main findings are as follows. First, as the tea industries are at different stages of development, compared with agricultural and non-agricultural part-time households, the welfare level of pure agricultural households is lowest in the ATTCS, while welfare is the highest in the FWTCS. Second, factors such as the area of tea gardens and the number of laborers significantly affect farmers' livelihood strategies transformation from pure agricultural households to agricultural part-time households in the ATTCS and FWTCS. Third, the high commodity rate of tea products, combined with compound cultivation in tea gardens, provides local people with essential sources of income, food, and nutrients, so as to improve food security in the ATTCS and FWTCS. These findings are essential for designing policies to ensure farmers' livelihoods and food security through AHSs and other sustainable agriculture.
RESUMO
Cytidine triphosphate synthase (CTPS) plays a pivotal role in the de novo synthesis of cytidine triphosphate (CTP), a fundamental building block for RNA and DNA that is essential for life. CTPS is capable of directly binding to all four nucleotide triphosphates: adenine triphosphate, uridine triphosphate, CTP, and guanidine triphosphate. Furthermore, CTPS can form cytoophidia in vivo and metabolic filaments in vitro, undergoing regulation at multiple levels. CTPS is considered a potential therapeutic target for combating invasions or infections by viral or prokaryotic pathogens. Utilizing cryo-electron microscopy, we determined the structure of Escherichia coli CTPS (ecCTPS) filament in complex with CTP, nicotinamide adenine dinucleotide (NADH), and the covalent inhibitor 6-diazo-5-oxo- l-norleucine (DON), achieving a resolution of 2.9 Å. We constructed a phylogenetic tree based on differences in filament-forming interfaces and designed a variant to validate our hypothesis, providing an evolutionary perspective on CTPS filament formation. Our computational analysis revealed a solvent-accessible ammonia tunnel upon DON binding. Through comparative structural analysis, we discern a distinct mode of CTP binding of ecCTPS that differs from eukaryotic counterparts. Combining biochemical assays and structural analysis, we determined and validated the synergistic inhibitory effects of CTP with NADH or adenine on CTPS. Our results expand our comprehension of the diverse regulatory aspects of CTPS and lay a foundation for the design of specific inhibitors targeting prokaryotic CTPS.
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Garnet-based electrolytes with high ionic conductivity and excellent stability against lithium metal anodes are promising for commercial applications in solid-state lithium batteries (SSLBs). However, the further development of SSLBs is inhibited by issues such as low ionic conductivity and uncontrolled lithium dendrite growth. Herein, we report the synthesis of fluorine-doped Li7La3Zr2O12 (LLZO-F0.2) fibers by electrospinning and the subsequent calcination at high temperatures. The solid composite electrolyte with LLZO-F0.2 exhibits an ionic conductivity of 5.37 × 10-4 S cm-1 and a high lithium-ion transference number of 0.61 at room temperature. Meanwhile, it exhibits lower resistance and more uniform lithium metal stripping and deposition in symmetric cells. The full cell with LiFePO4 cathode exhibits excellent rate capability and cycling stability for 800 cycles at 0.5 C with a discharge specific capacity retention of 97.7%. This fluorine-doped fibrous garnet-type electrolyte provides a viable option for preparing high-performance SSLBs.
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The real-time detection of intracellular biological processes by encoded sensors has broad application prospects. Here, we developed a degron-based modular reporting system, the Device of Death Operation (DODO), that can monitor various biological processes. The DODO system consists of a "reporter", an "inductor", and a "degron". After zymogen activation and cleavage, the degron will be released from the "reporter", which eventually leads to the stabilization of the "reporter", and can be detected. By replacing different "inductors" and "reporters", a series of biological processes can be reported through various signals. The system can effectively report the existence of TEV protease. To prove this concept, we successfully applied the DODO system to report apoptosis in 2D and 3D cultures. In addition, the reporter based on degron will help to design protease reporters other than caspase.
Assuntos
Apoptose , Humanos , Técnicas de Cultura de Células/métodos , Técnicas de Cultura de Células/instrumentaçãoRESUMO
Biochar and organic fertilizer are commonly used to maintain soil health and sustainable agroecosystems, and the alternate wet-dry management of soil moisture in dry direct-seeded paddy fields can complicate the effects of biochar and organic fertilizer on soil microhabitats. Therefore, this study used chicken manure organic fertilizer to replace some of the inorganic fertilizer and applied biochar to explore the ability of biochar and organic fertilizer to regulate the functions of the soil microhabitat in dry direct-seeded paddy fields. The coupling effect of organic fertilizer and biochar increased the diversity and richness of soil bacteria but had no significant effect on soil fungi. Biochar and organic fertilizer affected the distribution and composition of soil bacteria and fungi, and the total number of soil bacteria and fungi increased by 1365 and -71 (5 t/hm2 biochar and no organic fertilizer), 660 and 79 (10 t/hm2 biochar and no organic fertilizer), 3121 and 7 (no biochar and 20 % organic fertilizer substitution), 1873 and -72 (5 t/hm2 biochar and 20 % organic fertilizer substitution), and -544 and -65 (10 t/hm2 biochar and 20 % organic fertilizer substitution), respectively, compared with that of the control treatment. Compared with the application of biochar alone, the coupling effect of biochar and organic fertilizer increased the average degree (0.95 and 0.16), links (190 and 32), and ratio of fungal positive links (1.651 %), and decreased the modularity (0.034 and 0.052) and ratio of bacterial positive links (6.482 %) of bacterial and fungal networks. In addition, the coupling effect resulted in a more complex association between soil microbial diversity and richness and microbial ecological functions. Random forest predictions indicated that, organic fertilizer as a random factor, changes in the abundance of bacterial Bacteroidetes and Nitrospirae and fungal Monoblepharomycota were the main factors driving the differences in soil microbial ecological functions.
Assuntos
Agricultura , Carvão Vegetal , Fertilizantes , Microbiologia do Solo , Fertilizantes/análise , Agricultura/métodos , Solo/química , Bactérias , Fungos , Oryza/crescimento & desenvolvimento , EstercoRESUMO
The cellular compartmentation induced by self-assembly of natural proteins has recently attracted widespread attention due to its structural-functional significance. Among them, as a highly conserved metabolic enzyme and one of the potential targets for cancers and parasitic diseases in drug development, CTP synthase (CTPS) has also been reported to self-assemble into filamentous structures termed cytoophidia. To elucidate the dynamical mechanism of cytoophidium filamentation, we utilize single-molecule fluorescence imaging to observe the real-time self-assembly dynamics of CTPS and the coordinated assembly between CTPS and its interaction partner, Δ1-pyrroline-5-carboxylate synthase (P5CS). Significant differences exist in the direction of growth and extension when the two proteins self-assemble. The oligomer state distribution analysis of the CTPS minimum structural subunit under different conditions and the stoichiometry statistics of binding CTPS and P5CS by single-molecule fluorescence photobleach counting further confirm that the CTPS cytoophidia are mainly stacked with tetramers. CTPS can act as the nucleation core to induce the subsequent growth of the P5CS filaments. Our work not only provide evidence from the molecular level for the self-assembly and coordinated assembly (coassembly) of CTPS with its interaction partner P5CS in vitro but also offer new experimental perspectives for the dynamics research of coordinated regulation between other protein polymers.
Assuntos
Citoesqueleto , Ornitina-Oxo-Ácido Transaminase , Ornitina-Oxo-Ácido Transaminase/metabolismo , Citoesqueleto/metabolismo , Imagem ÓpticaRESUMO
In plants, the rapid accumulation of proline is a common response to combat abiotic stress1-7. Delta-1-pyrroline-5-carboxylate synthase (P5CS) is a rate-limiting enzyme in proline synthesis, catalysing the initial two-step conversion from glutamate to proline8. Here we determine the first structure of plant P5CS. Our results show that Arabidopsis thaliana P5CS1 (AtP5CS1) and P5CS2 (AtP5CS2) can form enzymatic filaments in a substrate-sensitive manner. The destruction of AtP5CS filaments by mutagenesis leads to a significant reduction in enzymatic activity. Furthermore, separate activity tests on two domains reveal that filament-based substrate channelling is essential for maintaining the high catalytic efficiency of AtP5CS. Our study demonstrates the unique mechanism for the efficient catalysis of AtP5CS, shedding light on the intricate mechanisms underlying plant proline metabolism and stress response.
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Proteínas de Arabidopsis , Arabidopsis , Arabidopsis/metabolismo , Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Prolina/metabolismo , Complexos Multienzimáticos , Fosfotransferases (Aceptor do Grupo Álcool) , Glutamato-5-Semialdeído DesidrogenaseRESUMO
The final step in the de novo synthesis of cytidine 5'-triphosphate (CTP) is catalyzed by CTP synthase (CTPS), which can form cytoophidia in all three domains of life. Recently, we have discovered that CTPS binds to ribonucleotides (NTPs) to form filaments, and have successfully solved the structures of Drosophila melanogaster CTPS bound with NTPs. Previous biochemical studies have shown that CTPS can bind to deoxyribonucleotides (dNTPs) to produce 2'-deoxycytidine-5'-triphosphate (dCTP). However, the structural basis of CTPS binding to dNTPs is still unclear. In this study, we find that Drosophila CTPS can also form filaments with dNTPs. Using cryo-electron microscopy, we are able to solve the structure of Drosophila melanogaster CTPS bound to dNTPs with a resolution of up to 2.7 Å. By combining these structural findings with biochemical analysis, we compare the binding and reaction characteristics of NTPs and dNTPs with CTPS. Our results indicate that the same enzyme can act bifunctionally as CTP/dCTP synthase in vitro, and provide a structural basis for these activities.
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Liquidâliquid phase separation (LLPS) is a ubiquitous process in which proteins, RNA, and biomolecules assemble into membrane-less compartments, playing important roles in many biological functions and diseases. The current knowledge on the biophysical and biochemical principles of LLPS is largely from in vitro studies; however, the physiological environment in living cells is complex and not at equilibrium. The characteristics of intracellular dynamics and their roles in physiological LLPS remain to be resolved. Here, by using single-particle tracking of quantum dots and dynamic monitoring of the formation of stress granules (SGs) in single cells, the spatiotemporal dynamics of intracellular transport in cells undergoing LLPS are quantified. It is shown that intracellular diffusion and active transport are both reduced. Furthermore, the formation of SG droplets contributes to increased spatial heterogeneity within the cell. More importantly, the study demonstrated that the LLPS of SGs can be regulated by intracellular dynamics in two stages: the reduced intracellular diffusion promotes SG assembly and the microtubule-associated transport facilitates SG coalescences. The work on intracellular dynamics not only improves the understanding of the mechanism of physiology phase separations occurring in nonequilibrium environments but also reveals an interplay between intracellular dynamics and LLPS.
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Pontos Quânticos , Humanos , Pontos Quânticos/metabolismo , Transporte Biológico/fisiologia , Grânulos de Estresse/metabolismo , Separação de FasesRESUMO
Oral microorganisms are closely related to oral health, the occurrence of some oral diseases is associated with changes in the oral microbiota, and many studies have demonstrated that traditional smoking can affect the oral microbial community. However, due to the short time since the emergence of e-cigarettes, fewer studies are comparing oral microorganisms for users of e-cigarettes versus cigarettes. We collected saliva from 40 non-smokers (NS), 46 traditional cigarette smokers (TS), and 27 e-cigarette consumers (EC), aged between 18 and 35 years. We performed 16S rRNA gene sequencing on the saliva samples collected to study the effects of e-cigarettes versus traditional cigarettes on the oral microbiome. The results showed that compared with the NS group, the alpha diversity of oral flora in saliva was altered in the TS group, with no significant change in the e-cigarette group. Compared with the NS and EC groups, the relative abundance of Actinomyces and Prevotella was increased in the TS group. However, compared with the NS and TS groups, the relative abundance of Veillonella was increased, and the relative abundance of Porphyromonas and Peptostreptococcus was decreased in the EC group. These results showed that both e-cigarettes and traditional cigarettes could alter the structure and composition of oral microbiota. The use of traditional cigarettes promotes the growth of some anaerobic bacteria, which may contribute to dental decay and bad breath over time. E-cigarettes have a different effect on the structure and composition of the oral microbial community compared to conventional cigarettes. In order to better understand the effects of e-cigarettes and traditional cigarettes on users' mouths, future studies will investigate the relationship between diseases such as dental caries and periodontitis and changes in oral microbial species levels.
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Nuclear factor erythroid 2-related factor-2 (Nrf2) antioxidant signaling is involved in liver protection, but this generalization overlooks conflicting studies indicating that Nrf2 effects are not necessarily hepatoprotective. The role of Nrf2/heme oxygenase-1 (HO-1) in cholestatic liver injury (CLI) remains poorly defined. Here, we report that Nrf2/HO-1 activation exacerbates liver injury rather than exerting a protective effect in CLI. Inhibiting HO-1 or ameliorating bilirubin transport alleviates liver injury in CLI models. Nrf2 knockout confers hepatoprotection in CLI mice, whereas in non-CLI mice, Nrf2 knockout aggravates liver damage. In the CLI setting, oxidative stress activates Nrf2/HO-1, leads to bilirubin accumulation, and impairs mitochondrial function. High levels of bilirubin reciprocally upregulate the activation of Nrf2 and HO-1, while antioxidant and mitochondrial-targeted SOD2 overexpression attenuate bilirubin toxicity. The expression of Nrf2 and HO-1 is elevated in serum of patients with CLI. These results reveal an unrecognized function of Nrf2 signaling in exacerbating liver injury in cholestatic disease.