RESUMO
Cellular processes are subject to inherent variability, but the extent to which cells can regulate this variability has received little investigation. Here, we explore the characteristics of the rate of cellular protein synthesis in single cells of the eukaryote fission yeast. Strikingly, this rate is highly variable despite protein synthesis being dependent on hundreds of reactions which might be expected to average out at the overall cellular level. The rate is variable over short time scales, and exhibits homoeostatic behaviour at the population level. Cells can regulate the level of variability through processes involving the TOR pathway, suggesting there is an optimal level of variability conferring a selective advantage. While this could be an example of bet-hedging, but we propose an alternative explanation: regulated 'loose' control of complex processes of overall cellular metabolism such as protein synthesis, may lead to this variability. This could ensure cells are fluid in control and agile in response to changing conditions, and may constitute a novel organisational principle of complex metabolic cellular systems.
Assuntos
Biossíntese de Proteínas , SchizosaccharomycesRESUMO
Introns are ubiquitous in eukaryotic transcripts. They are often viewed as junk RNA but the huge energetic burden of transcribing, removing, and degrading them suggests a significant evolutionary advantage. Ostensibly, an intron functions within the host pre-mRNA to regulate its splicing, transport, and degradation. However, recent studies have revealed an entirely new class of trans-acting functions where the presence of intronic RNA in the cell impacts the expression of other genes in trans. Here, we review possible new mechanisms of intron functions, with a focus on the role of yeast introns in regulating the cell growth response to starvation.
Assuntos
Genoma , Genômica , Íntrons , Animais , Células Eucarióticas/metabolismo , Evolução Molecular , Expressão Gênica , Regulação da Expressão Gênica , Genômica/métodos , Humanos , Precursores de RNA , Splicing de RNA , Estabilidade de RNA , Leveduras/genéticaRESUMO
Plants can adjust their growth to specific times of the day and season. Different photoperiods result in distinct growth patterns, which correlate with specific carbon-partitioning strategies in source (leaves) and sink (roots) organs. Therefore, external cues such as light, day length, and temperature need to be integrated with intracellular processes controlling overall carbon availability and anabolism. The target of rapamycin (TOR) pathway is a signalling hub where environmental signals, circadian information, and metabolic processes converge to regulate plant growth. TOR complex mutants display altered patterns of root growth and starch levels. Moreover, depletion of TOR or reduction in cellular energy levels affect the pace of the clock by extending the period length, suggesting that this pathway could participate in circadian metabolic entrainment. However, this seems to be a mutual interaction, since the TOR pathway components are also under circadian regulation. These results strengthen the role of this signalling pathway as a master sensor of metabolic status, integrating day length and circadian cues to control anabolic processes in the cell, thus promoting plant growth and development. Expanding this knowledge from Arabidopsis thaliana to crops will improve our understanding of the molecular links connecting environmental perception and growth regulation under field conditions.
Assuntos
Proteínas de Arabidopsis , Arabidopsis , Relógios Circadianos , Fotoperíodo , Proteínas de Arabidopsis/metabolismo , Relógios Circadianos/fisiologia , Regulação da Expressão Gênica de Plantas , Sirolimo/metabolismo , Arabidopsis/metabolismo , Carbono/metabolismo , Ritmo Circadiano/fisiologia , Fosfatidilinositol 3-Quinases/metabolismoRESUMO
Altered metabolism is a hallmark of aging. The tricarboxylic acid cycle (TCA cycle) is an essential metabolic pathway and plays an important role in lifespan regulation. Supplementation of α-ketoglutarate, a metabolite converted by isocitrate dehydrogenase alpha-1 (idha-1) in the TCA cycle, increases lifespan in C. elegans. However, whether idha-1 can regulate lifespan in C. elegans remains unknown. Here, we reported that the expression of idha-1 modulates lifespan and oxidative stress tolerance in C. elegans. Transgenic overexpression of idha-1 extends lifespan, increases the levels of NADPH/NADP+ ratio, and elevates the tolerance to oxidative stress. Conversely, RNAi knockdown of idha-1 exhibits the opposite effects. In addition, the longevity of eat-2 (ad1116) mutant via dietary restriction (DR) was reduced by idha-1 knockdown, indicating that idha-1 may play a role in DR-mediated longevity. Furthermore, idha-1 mediated lifespan may depend on the target of rapamycin (TOR) signaling. Moreover, the phosphorylation levels of S6 kinase (p-S6K) inversely correlate with idha-1 expression, supporting that the idha-1-mediated lifespan regulation may involve the TOR signaling pathway. Together, our data provide new insights into the understanding of idha-1 new function in lifespan regulation probably via DR and TOR signaling and in oxidative stress tolerance in C. elegans.
Assuntos
Proteínas de Caenorhabditis elegans , Caenorhabditis elegans , Isocitrato Desidrogenase , Longevidade , Estresse Oxidativo , Animais , Caenorhabditis elegans/enzimologia , Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/metabolismo , Isocitrato Desidrogenase/genética , Isocitrato Desidrogenase/metabolismo , Longevidade/genéticaRESUMO
The Estrogen-related receptor (ERR) can regulate the growth and development, metabolism, reproduction, and other physiological activities of insects, but its specific mechanism of action is still unclear. The aim of this study was to explore the relationship between expression of ERR and Vitellogenins (Vg) and the juvenile hormone (JH) and insulin/insulin-like growth factor/target of rapamycin (IIS/TOR) signaling pathways in Polyrhachis vicina Roger. P. vicina was used as the experimental model to clone the PvVg gene, perform double-stranded RNA synthesis and delivery and observe the effects of pharmacological treatments. The full-length PvVg cDNA product is 5586 bp. Higher PvVg mRNA expression was seen in the pupa and adults, and varying levels were seen in the different body parts of three different castes. RNA interference of PvVg expression led to disturbed development, an abnormal phenotype, and high mortality. PvVg RNAi also led to a reduction in mRNA levels of PvERR, ultraspiracle (PvUSP), forkhead box protein O (PvFOXO) and PvTOR genes in fourth instar larval, but a significant increase was seen in pupa and females. No significant change was seen in workers and males. After PvVg knockdown, application of exogenous JHIII reduced the expression of these genes in pupa and females, increased expression in workers, and decreased PvUSP mRNA expression in males. Both protein and mRNA expression levels of PvFOXO were affected by PvVg RNAi. PvERR RNAi increased PvVg expression in pupa and females and Kruppel-homolog 1 (PvKr-h1) and PvFOXO expression in males. The results of this study suggest that there is an interaction between PvERR and PvVg, and that crosstalk with the JH and IIS/TOR signaling pathways can affect development and reproduction. This effect is caste and developmental stage specific. We also speculate that the FOXO/USP complex participates in JH regulation of PvVg in P. vicina.
Assuntos
Formigas , Hormônios Juvenis , Animais , Formigas/genética , Estrogênios , Feminino , Proteínas de Insetos/genética , Proteínas de Insetos/metabolismo , Hormônios Juvenis/metabolismo , Masculino , Interferência de RNA , Receptores de Estrogênio/metabolismo , Transdução de Sinais , Vitelogeninas/genética , Vitelogeninas/metabolismoRESUMO
The purpose of this study was to explore the protective effect of BW373U86 (a δ-opioid receptor (DOR) agonist) on ischemia-reperfusion (I/R) injury in rat cardiomyocytes and its underlying mechanism. Primary rat cardiomyocytes were cultured and pretreated with BW373U86 for intervention. The cardiomyocytes were cultured under the condition of 94% N2 and 5% CO2 for 24 h to perform hypoxia culture and conventionally cultured for 12 h to perform reoxygenation culture. The cell viability of cardiomyocytes was detected by an MTT assay (Sigma-Aldrich). The autophagy lysosome levels in cardiomyocytes were evaluated by acidic vesicular organelles with dansylcadaverine (MDC) staining (autophagy test kit, Kaiji Biology, kgatg001). The protein expression levels of LC3, p62, and factors in the PI3K/Akt/mTOR signaling pathway were detected by Western blot. Pretreatment with BW373U86 could improve the cell viability of cardiomyocytes with hypoxia-reoxygenation (H/R) injury (p < 0.05). Interestingly, after coculture of BW373U86 and PI3K inhibitor (3-methyladenine), the protein expression levels of p-Akt in cardiomyocytes were markedly increased in comparison with those in the BW373U86 group (p < 0.05). However, there were no significant differences in the protein expression levels of mTOR between the coculture group and the BW373U86 group (p > 0.05). BW373U86 upregulated autophagy to protect cardiomyocytes from H/R injury, which may be related to the PI3K/Akt/m TOR pathway.
Assuntos
Autofagia/efeitos dos fármacos , Benzamidas/farmacologia , Traumatismo por Reperfusão Miocárdica/prevenção & controle , Miócitos Cardíacos/efeitos dos fármacos , Fosfatidilinositol 3-Quinases/metabolismo , Piperazinas/farmacologia , Proteínas Proto-Oncogênicas c-akt/metabolismo , Receptores Opioides delta/agonistas , Serina-Treonina Quinases TOR/metabolismo , Animais , Hipóxia Celular , Células Cultivadas , Traumatismo por Reperfusão Miocárdica/enzimologia , Traumatismo por Reperfusão Miocárdica/patologia , Miócitos Cardíacos/enzimologia , Miócitos Cardíacos/patologia , Fosforilação , Ratos , Ratos Sprague-Dawley , Receptores Opioides delta/metabolismo , Transdução de SinaisRESUMO
In order to illustrate the immunometabolic changes of fish during bacterial infection, grass carp (Ctenopharyngodon idellus) was injected with Flavobacteriumcolumnare(F.columnare) and then the immune response, nutrient metabolism and related signaling pathways were assayed from 6â¯h post injection (hpi) to 7 days post injection (dpi). After F.columnare injection, gill lamellae showed obvious fusion and higher mRNA expression levels of pro-inflammatory cytokines. The mRNA expression levels of TNF-α, IL-1ß and IL-8 in the head kidney were also significantly upregulated at 6 hpi and 3 dpi. Moreover, the expression of IgZ in the gill was significantly upregulated at 3 dpi and 7 dpi, while the expression of IgM in the head kidney was significantly upregulated at 1 dpi and 3 dpi after F.columnare injection. During bacterial infection, the systematic nutrient metabolism was also significantly affected. Hepatic glycolysis, indicated by GK mRNA expression and PK activity, was significantly upregulated at 1 dpi, while glucogenesis, indicated by PEPCK mRNA expression and enzyme activity, was significantly increased at later time, which resulted in the decreased hepatic glycogen content at 1dpi but increased glycogen content at 7 dpi in the experimental group. LPL, which catalyzed the lipid catabolism, showed decreased mRNA expression and enzyme activity at 6 hpi, while ACC, which was rate-limiting of FA synthesis, was significantly increased at 6 hpi, 3 dpi and 7 dpi. During this process, the nutrient sensing signaling was also significantly affected. TOR signaling in grass carp was significantly activated while ERK signaling was significantly inhibited after F.columnare infection, both of which might function as the sensor and regulator of fish immunometabolic changes.
Assuntos
Carpas/imunologia , Doenças dos Peixes/imunologia , Infecções por Flavobacteriaceae/veterinária , Imunidade Inata/genética , Nutrientes/metabolismo , Transdução de Sinais , Animais , Carpas/genética , Carpas/metabolismo , Infecções por Flavobacteriaceae/imunologia , Flavobacterium/fisiologiaRESUMO
Soybean saponin (SA) is known as a major anti-nutritional factor that causes metabolic disturbances and growth reduction in fish. However, the mechanisms underlying these effects were far from fully understood. In particular, the influences of SA on nutrient sensing and downstream metabolic pathways remain largely unexplored. Using zebrafish as an animal model, this study was conducted to examine the phenotypic and molecular responses after dietary SA treatment for 2â¯weeks. SA at both 5 and 10â¯g/kg diet levels significantly reduced growth performance and feed efficiency, and damaged the morphology of the intestinal mucosa. SA stimulated AMP-activated protein kinase but reduced target of rapamycin (TOR) activities in both feeding trial and cellular studies. Furthermore, SA increased the mRNA expressions of growth axis genes including growth hormone, insulin-like growth factor 1, growth hormone receptor A, and growth hormone receptor B, but decreased insulin-like growth factor-binding protein 2 at both mRNA and protein levels. SA also increased the expressions of key metabolic enzymes involved in glutamine synthetase, glutamate dehydrogenase and lipolysis, hormone-sensitive lipase and lipoprotein lipase. Our results demonstrated that SA modulated nutrient sensing pathways and metabolism, thus provide new aspects on the explanation of the physiological effects of SA.
Assuntos
Glycine max/metabolismo , Saponinas/metabolismo , Peixe-Zebra , AnimaisRESUMO
Bud27 and its human orthologue URI (unconventional prefoldin RPB5-interactor) are members of the prefoldin (PFD) family of ATP-independent molecular chaperones binding the Rpb5 subunit to all three nuclear eukaryotic RNA polymerases (RNA pols). Bud27/URI are considered to function as a scaffold protein able to assemble additional members of the prefoldin (PDF) family in both human and yeast. Bud27 and URI are not subunits of the canonical PFD/GimC complex and not only the composition but also other functions independent of the PFD/GimC complex have been described for Bud27 and URI. Bud27 interacts only with Pfd6 but no other components of the R2TP/PFDL. Furthermore previously reported interaction between Bud27 and Pfd2 was not later confirmed. These results point to major differences in the prefoldin-like complex composition between yeast and other organisms, suggesting also important differences in functions. Furthermore, this assumption could be extended to the R2TP/PFDL complex, which has been shown to differ between different organisms and has not been identified in yeast. This casts doubt on whether Bud27 cooperation with prefoldin and other components of the R2TP/PFDL modules are required for its action. This could be extended to URI and point to a role of Bud27/URI in cell functions more relevant than this previously proposed as co-prefoldin.
Assuntos
Chaperonas Moleculares/química , Fatores de Iniciação de Peptídeos/química , Proteínas de Saccharomyces cerevisiae/química , Saccharomyces cerevisiae , RNA Polimerases Dirigidas por DNARESUMO
To investigate the inhibitory effect of isobutyrylshikonin on the growth of human colon carcinoma cells in vitro and its effect on the PI3K/Akt/m-TOR pathway. MTT assay was used to detect the inhibitory effect of different concentrations (0, 6.25, 12.5, 25, 50, 100 mg·L⻹) of isobutyrylshikonin on the proliferation of human colon carcinoma cell HT29 at 24, 48 h. CCK-8 method was used to detect the inhibitory effect of isobutyrylshikonin on HT29, HCT116, DLD-1 and Caco-2 at 48 h. AnnexinV/propidium iodide staining was applied in detecting the apoptoticrate of HT29 cells treated with different concentrations of isobutyrylshikonin at 24 h and 48 h. Cycletest plus DNA was employed to analyze HT29 apoptosis and cell cycle after 48 h treatment with isobutyrylshikonin at different concentrations. Western blot and RT-PCR assay were used to examine the protein and mRNA expressions of PI3K, p-PI3K, Akt, p-Akt and m-TOR. The results showed that isobutyrylshikonin inhibited the proliferation of different human colon carcinoma cells, and the inhibition rate was in a dose-dependent manner. Isobutyrylshikonin induced apoptosis mainly in the early stage and blocked cells in the G0/G1 or G2/M phase. Isobutyrylshikonin reduced the protein expressions of PI3K, p-PI3K, Akt, p-Akt, m-TOR and the mRNA expressions of PI3K, Akt, m-TOR in a dose-dependent manner. Isobutyrylshikonin can significantly inhibit the proliferation, induce the early apoptosis and change the cycle distribution in colon carcinoma cells.This biological effect may be correlated with the inhibition of PI3K/AKT/m-TOR pathway.
Assuntos
Proliferação de Células , Naftoquinonas/farmacologia , Transdução de Sinais , Apoptose , Células CACO-2 , Ciclo Celular , Células HT29 , Humanos , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Serina-Treonina Quinases TOR/metabolismoRESUMO
Environmental changes, such as nutrient limitation or starvation induce different signal transducing pathways, which require coordinated cooperation of several genes. Our previous data revealed that the fhl1 fork-head type transcription factor of the fission yeast could be involved in sporulation, which was typically induced under poor conditions. Since the exact role of Fhl1 in this process was not known, we wanted to identify its downstream targets and to investigate its possible cooperation with another known regulator of sporulation. Gene expression and Northern blot analysis of the fhl1∆ mutant strain revealed the target genes involved in mating and sporulation. Our results also showed that Fhl1 could regulate nutrient sensing, the transporter and permease genes. Since the majority of these genes belonged to the nitrogen starvation response, the possible cooperation of fhl1 and tor2 was also investigated. Comparison of their microarray data and the expression of fhl1 + from a strong promoter in the tor2-ts mutant cells suggested that one part of the target genes are commonly regulated by Fhl1 and Tor2. Since the expression of fhl1 + from a strong promoter could rescue rapamycin and temperature sensitivity and suppressed the hyper-sporulation defect of the tor2-ts mutant cells, we believe that Fhl1 acts in TOR signaling, downstream of Tor2. Thus, this work shed light on certain novel details of the regulation of the sexual processes and a new member of the TOR pathway, but further experiments are needed to confirm the involvement of Fhl1 in nutrient sensing.
Assuntos
Fatores de Transcrição Forkhead/genética , Regulação Fúngica da Expressão Gênica , Meiose/genética , Complexos Multiproteicos/metabolismo , Nitrogênio/metabolismo , Proteínas de Schizosaccharomyces pombe/genética , Schizosaccharomyces/genética , Schizosaccharomyces/metabolismo , Transdução de Sinais , Serina-Treonina Quinases TOR/metabolismo , Análise por Conglomerados , Biologia Computacional/métodos , Perfilação da Expressão Gênica , Estudo de Associação Genômica Ampla , Alvo Mecanístico do Complexo 1 de Rapamicina , Anotação de Sequência Molecular , Mutação , Regiões Promotoras Genéticas , Esporos Fúngicos/genéticaRESUMO
Two nutrient-controlled signalling pathways, the PKA and TOR pathway, play a major role in nutrient regulation of growth as well as growth-correlated properties in yeast. The relationship between the two pathways is not well understood. We have used Gap1 and Pho84 transceptor-mediated activation of trehalase and phosphorylation of fragmented Sch9 as a read-out for rapid nutrient activation of PKA or TORC1, respectively. We have identified conditions in which L-citrulline-induced activation of Sch9 phosphorylation is compromised, but not activation of trehalase: addition of the TORC1 inhibitor, rapamycin and low levels of L-citrulline. The same disconnection was observed for phosphate activation in phosphate-starved cells. The leu2 auxotrophic mutation reduces amino acid activation of trehalase, which is counteracted by deletion of GCN2. Both effects were also independent of TORC1. Our results show that rapid activation of the TOR pathway by amino acids is not involved in rapid activation of the PKA pathway and that effects of Gcn2 inactivation as well as leu2 auxotrophy all act independently of the TOR pathway. Hence, rapid nutrient signalling to PKA and TOR in cells arrested by nutrient starvation acts through parallel pathways.
Assuntos
Sistemas de Transporte de Aminoácidos/metabolismo , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Leucina/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Simportadores de Próton-Fosfato/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/enzimologia , Saccharomyces cerevisiae/metabolismo , Fosforilação , Processamento de Proteína Pós-Traducional , Transdução de Sinais , Fatores de Transcrição/metabolismoRESUMO
An early burst of hydrogen sulfide (H2S) produced by Saccharomyces cerevisiae during fermentation could increase varietal thiols and therefore enhance desirable tropical aromas in varieties such as Sauvignon Blanc. Here we attempted to identify genes affecting H2S formation from cysteine by screening yeast deletion libraries via a colony colour assay on media resembling grape juice. Both Δlst4 and Δlst7 formed lighter coloured colonies and produced significantly less H2S than the wild type on high concentrations of cysteine, likely because they are unable to take up cysteine efficiently. We then examined the nine known cysteine permeases and found that deletion of AGP1, GNP1 and MUP1 led to reduced production of H2S from cysteine. We further showed that deleting genes involved in the SPS-sensing pathway such as STP1 and DAL81 also reduced H2S from cysteine. Together, this study indirectly confirms that Agp1p, Gnp1p and Mup1p are the major cysteine permeases and that they are regulated by the SPS-sensing and target of rapamycin pathways under the grape juice-like, cysteine-supplemented, fermentation conditions. The findings highlight that cysteine transportation could be a limiting factor for yeast to generate H2S from cysteine, and therefore selecting wine yeasts without defects in cysteine uptake could maximise thiol production potential.
Assuntos
Cisteína/metabolismo , Sulfeto de Hidrogênio/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Transporte Biológico , Fermentação , Deleção de Genes , Testes Genéticos , Proteínas de Saccharomyces cerevisiae/genéticaRESUMO
The target of rapamycin (TOR) signaling pathway plays critical roles in controlling cell growth in a variety of eukaryotes. However, the contribution of this pathway in regulating virulence of plant pathogenic fungi is unknown. We identified and characterized nine genes encoding components of the TOR pathway in Fusarium graminearum. Biological, genetic and biochemical functions of each component were investigated. The FgFkbp12-rapamycin complex binds to the FgTor kinase. The type 2A phosphatases FgPp2A, FgSit4 and FgPpg1 were found to interact with FgTap42, a downstream component of FgTor. Among these, we determined that FgPp2A is likely to be essential for F. graminearum survival, and FgSit4 and FgPpg1 play important roles in cell wall integrity by positively regulating the phosphorylation of FgMgv1, a key MAP kinase in the cell wall integrity pathway. In addition, the FgPpg1 interacting protein, FgTip41, is involved in regulating mycelial growth and virulence. Notably, FgTip41 does not interact with FgTap42 but with FgPpg1, suggesting the existence of FgTap42:FgPpg1:FgTip41 heterotrimer in F. graminearum, a complex not observed in the yeast model. Collectively, we defined a genetic regulatory framework that elucidates how the TOR pathway regulates virulence and vegetative development in F. graminearum.
Assuntos
Fusarium/crescimento & desenvolvimento , Fusarium/patogenicidade , Transdução de Sinais , Serina-Treonina Quinases TOR/metabolismo , Virulência , Farmacorresistência Fúngica/genética , Fusarium/genética , Regulação Fúngica da Expressão Gênica , Genes Fúngicos , Teste de Complementação Genética , Saccharomyces cerevisiae , Deleção de Sequência , Sirolimo/farmacologia , Serina-Treonina Quinases TOR/genética , Proteína 1A de Ligação a Tacrolimo/genética , Proteína 1A de Ligação a Tacrolimo/metabolismo , Tricotecenos/metabolismo , Técnicas do Sistema de Duplo-HíbridoRESUMO
The three closely related groups of serine/threonine protein phosphatases PP2A, PP4 and PP6 are conserved throughout eukaryotes. The catalytic subunits are present in trimeric and dimeric complexes with scaffolding and regulatory subunits that control activity and confer substrate specificity to the protein phosphatases. In Arabidopsis, three scaffolding (A subunits) and 17 regulatory (B subunits) proteins form complexes with five PP2A catalytic subunits giving up to 255 possible combinations. Three SAP-domain proteins act as regulatory subunits of PP6. Based on sequence similarities with proteins in yeast and mammals, two putative PP4 regulatory subunits are recognized in Arabidopsis. Recent breakthroughs have been made concerning the functions of some of the PP2A and PP6 regulatory subunits, for example the FASS/TON2 in regulation of the cellular skeleton, B' subunits in brassinosteroid signalling and SAL proteins in regulation of auxin transport. Reverse genetics is starting to reveal also many more physiological functions of other subunits. A system with key regulatory proteins (TAP46, TIP41, PTPA, LCMT1, PME-1) is present in all eukaryotes to stabilize, activate and inactivate the catalytic subunits. In this review, we present the status of knowledge concerning physiological functions of PP2A, PP4 and PP6 in Arabidopsis, and relate these to yeast and mammals.
Assuntos
Meio Ambiente , Fosfoproteínas Fosfatases/metabolismo , Desenvolvimento Vegetal , Plantas/enzimologia , Animais , Domínio Catalítico , Saccharomyces cerevisiae/enzimologiaRESUMO
The PAR-domain protein 1 (PDP1) is essential for locomotor activity of insects. However, its functions in insect growth and development have not been studied extensively, which prompted our hypothesis that PDP1 acts in energy metabolism. Here we report identification of TcPDP1 in the red flour beetle, Tribolium castaneum, and its functional analysis by RNAi. Treating larvae with dsTcPDP1 induced pupae developmental arrestment, accompanied by accelerated fat body degradation. dsTcPDP1 treatments in adults resulted in reduced female fecundity. Disruption of TcPDP1 expression affected the transcription of genes involved in insulin signaling transduction and mechanistic target of rapamycin (mTOR) pathway. These results support our hypothesis that TcPDP1 acts in energy metabolism in T. castaneum.
Assuntos
Fatores de Transcrição de Zíper de Leucina Básica/metabolismo , Metabolismo Energético/fisiologia , Proteínas de Insetos/metabolismo , Insulina/metabolismo , Serina-Treonina Quinases TOR/metabolismo , Tribolium/metabolismo , Animais , Fatores de Transcrição de Zíper de Leucina Básica/genética , Feminino , Regulação da Expressão Gênica no Desenvolvimento , Proteínas de Insetos/genética , Insulina/genética , Larva/genética , Larva/metabolismo , Masculino , Pupa/genética , Pupa/metabolismo , Transdução de Sinais , Serina-Treonina Quinases TOR/genética , Tribolium/genética , Tribolium/crescimento & desenvolvimentoRESUMO
Rice wine is a popular traditional alcoholic drink with a long history in China. However, the presence of the potential carcinogen ethyl carbamate (EC) raises a series of food safety concerns. Although the metabolic pathway of urea (the major precusor of EC) has been characterized in Saccharomyces cerevisiae, the regulation of urea accumulation remains unclear, making the efficient elimination of urea difficult. To demonstrate the regulatory mechanisms governing urea accumulation, three key nitrogen sources that can inhibit urea utilization for a commercial S. cerevisiae strain were identified. In addition, regulators of nitrogen catabolite repression (NCR) and target of rapamycin (TOR) pathways were identified as being involved in urea accumulation by real-time quantitative PCR. Based on these results, preferred nitrogen sources were found to repress urea utilization by converting them to glutamine or glutamate. Moreover, the results indicated that the manner of urea metabolism regulation was different for two positive regulators involved in NCR; Gln3p can be retained in the cytoplasm by glutamine, while Gat1p can be retained by glutamine and glutamate. Furthermore, this was confirmed by fluorescence location detection. These new findings provide new targets for eliminating EC and other harmful nitrogen-containing compounds in fermented foods.
Assuntos
Nitrogênio/metabolismo , Saccharomyces cerevisiae/metabolismo , Ureia/metabolismo , Repressão Catabólica , Regulação Fúngica da Expressão Gênica , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismoRESUMO
Ribosomes are the basis for protein production, whose biogenesis is essential for cells to drive growth and proliferation. Ribosome biogenesis is highly regulated in accordance with cellular energy status and stress signals. In eukaryotic cells, response to stress signals and the production of newly-synthesized ribosomes require elements to be transcribed by the three RNA polymerases (RNA pols). Thus, cells need the tight coordination of RNA pols to adjust adequate components production for ribosome biogenesis which depends on environmental cues. This complex coordination probably occurs through a signaling pathway that links nutrient availability with transcription. Several pieces of evidence strongly support that the Target of Rapamycin (TOR) pathway, conserved among eukaryotes, influences the transcription of RNA pols through different mechanisms to ensure proper ribosome components production. This review summarizes the connection between TOR and regulatory elements for the transcription of each RNA pol in the budding yeast Saccharomyces cerevisiae. It also focuses on how TOR regulates transcription depending on external cues. Finally, it discusses the simultaneous coordination of the three RNA pols through common factors regulated by TOR and summarizes the most important similarities and differences between S. cerevisiae and mammals.
Assuntos
Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae , Animais , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Sirolimo/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , RNA Polimerases Dirigidas por DNA/metabolismo , Transdução de Sinais/fisiologia , RNA/metabolismo , Mamíferos/metabolismoRESUMO
Gene targeting is a commonly used method to reveal the function of genes. Although it is an attractive tool for molecular studies, it can frequently be a challenge because its efficiency can be low and it requires the screening of a large number of transformants. Generally, these problems originate from the elevated level of ectopic integration caused by non-homologous DNA end joining (NHEJ). To eliminate this problem, NHEJ-related genes are frequently deleted or disrupted. Although these manipulations can improve gene targeting, the phenotype of the mutant strains raised the question of whether mutations have side effects. The aim of this study was to disrupt the lig4 gene in the dimorphic fission yeast, S. japonicus, and investigate the phenotypic changes of the mutant strain. The mutant cells have shown various phenotypic changes, such as increased sporulation on complete medium, decreased hyphal growth, faster chronological aging, and higher sensitivity to heat shock, UV light, and caffeine. In addition, higher flocculation capacity has been observed, especially at lower sugar concentrations. These changes were supported by transcriptional profiling. Many genes belonging to metabolic and transport processes, cell division, or signaling had altered mRNA levels compared to the control strain. Although the disruption improved the gene targeting, we assume that the lig4 inactivation can cause unexpected physiological side effects, and we have to be very careful with the manipulations of the NHEJ-related genes. To reveal the exact mechanisms behind these changes, further investigations are required.
RESUMO
Restricting ribosome biosynthesis and assembly in response to nutrient starvation is a universal phenomenon that enables cells to survive with limited intracellular resources. When cells experience starvation, nutrient signaling pathways, such as the target of rapamycin (TOR) and protein kinase A (PKA), become quiescent, leading to several transcription factors and histone modification enzymes cooperatively and rapidly repressing ribosomal genes. Fission yeast has factors for heterochromatin formation similar to mammalian cells, such as H3K9 methyltransferase and HP1 protein, which are absent in budding yeast. However, limited studies on heterochromatinization in ribosomal genes have been conducted on fission yeast. Herein, we shed light on and compare the regulatory mechanisms of ribosomal gene transcription in two species with the latest insights.