RESUMO
BACKGROUND: Shoot branching is one of the important agronomic traits affecting yields and quality of tea plant (Camellia sinensis). Cytokinins (CTKs) play critical roles in regulating shoot branching. However, whether and how differently alternative splicing (AS) variant of CTKs-related genes can influence shoot branching of tea plant is still not fully elucidated. RESULTS: In this study, five AS variants of CTK biosynthetic gene adenylate isopentenyltransferase (CsA-IPT5) with different 3' untranslated region (3' UTR) and 5' UTR from tea plant were cloned and investigated for their regulatory effects. Transient expression assays showed that there were significant negative correlations between CsA-IPT5 protein expression, mRNA expression of CsA-IPT5 AS variants and the number of ATTTA motifs, respectively. Shoot branching processes induced by exogenous 6-BA or pruning were studied, where CsA-IPT5 was demonstrated to regulate protein synthesis of CsA-IPT5, as well as the biosynthesis of trans-zeatin (tZ)- and isopentenyladenine (iP)-CTKs, through transcriptionally changing ratios of its five AS variants in these processes. Furthermore, the 3' UTR AS variant 2 (3AS2) might act as the predominant AS transcript. CONCLUSIONS: Together, our results indicate that 3AS2 of the CsA-IPT5 gene is potential in regulating shoot branching of tea plant and provides a gene resource for improving the plant-type of woody plants.
Assuntos
Alquil e Aril Transferases/fisiologia , Camellia sinensis/enzimologia , Camellia sinensis/crescimento & desenvolvimento , Regiões 3' não Traduzidas , Alquil e Aril Transferases/genética , Camellia sinensis/genética , Clonagem Molecular , DNA de Plantas , Motivos de Nucleotídeos , Desenvolvimento Vegetal/genética , Brotos de Planta/genética , Brotos de Planta/crescimento & desenvolvimento , Análise de Sequência de DNARESUMO
Auxin is a key regulator of plant development affecting the formation and maturation of reproductive structures. The apoplastic route of auxin transport engages influx and efflux facilitators from the PIN, AUX and ABCB families. The polar localization of these proteins and constant recycling from the plasma membrane to endosomes is dependent on Rab-mediated vesicular traffic. Rab proteins are anchored to membranes via posttranslational addition of two geranylgeranyl moieties by the Rab Geranylgeranyl Transferase enzyme (RGT), which consists of RGTA, RGTB and REP subunits. Here, we present data showing that seed development in the rgtb1 mutant, with decreased vesicular transport capacity, is disturbed. Both pre- and post-fertilization events are affected, leading to a decrease in seed yield. Pollen tube recognition at the stigma and its guidance to the micropyle is compromised and the seed coat forms incorrectly. Excess auxin in the sporophytic tissues of the ovule in the rgtb1 plants leads to an increased tendency of autonomous endosperm formation in unfertilized ovules and influences embryo development in a maternal sporophytic manner. The results show the importance of vesicular traffic for sexual reproduction in flowering plants, and highlight RGTB1 as a key component of sporophytic-filial signaling.
Assuntos
Arabidopsis/enzimologia , Sementes/enzimologia , Transdução de Sinais , Alquil e Aril Transferases/metabolismo , Alquil e Aril Transferases/fisiologia , Arabidopsis/crescimento & desenvolvimento , Arabidopsis/metabolismo , Transporte Biológico , Regulação da Expressão Gênica de Plantas , Ácidos Indolacéticos/metabolismo , Mutação , Tubo Polínico/fisiologia , Sementes/crescimento & desenvolvimento , Sementes/metabolismoRESUMO
Pine wood nematode (PWN; Bursaphelenchus xylophilus), a destructive pest of Pinus massoniana, is causing a severe epidemic of pine wilt disease in China. When invaded by PWN, resistant P. massoniana secretes an abundance of oleoresin terpenoids as a defensive strategy. However, regulatory mechanisms of this defence in resistant P. massoniana have yet to be elucidated. Here, we characterized two terpene synthase genes, α-pinene synthase (PmTPS4) and longifolene synthase (PmTPS21), identified in resistant P. massoniana and investigate the contribution of these genes to the oleoresin defence strategy in resistant masson pines. Up-regulation of these two genes in the stem supported their involvement in terpene biosynthesis as part of the defence against PWN. Recombinant protein expression revealed catalytic activity for the two PmTPSs, with PmTPS4 primarily producing α-pinene, while PmTPS21 produced α-pinene and longifolene simultaneously. The major enzymatic products of the two terpene synthases had inhibitory effects on PWN in vitro. We demonstrated that PmTPS4 and PmTPS21 played positive roles in terpene-defence mechanisms against PWN infestation. The major products of these terpene synthases could directly inhibit the survival rate of PWN in vitro. We revealed that PmTPS21 was a novel bifunctional enzyme capable of simultaneous production of both monoterpene and sesquiterpene.
Assuntos
Alquil e Aril Transferases/metabolismo , Nematoides , Pinus/metabolismo , Defesa das Plantas contra Herbivoria , Proteínas de Plantas/metabolismo , Alquil e Aril Transferases/genética , Alquil e Aril Transferases/fisiologia , Animais , Deleção Clonal , Cromatografia Gasosa-Espectrometria de Massas , Filogenia , Pinus/genética , Pinus/fisiologia , Proteínas de Plantas/genética , Proteínas de Plantas/fisiologia , Reação em Cadeia da Polimerase em Tempo Real , Análise de Sequência de DNARESUMO
Two-component systems (TCS) in plants have evolved into a more complicated multi-step phosphorelay (MSP) pathway, which employs histidine kinases (HKs), histidine-containing phosphotransfer proteins (HPts), and response regulators (RRs) to regulate various aspects of plant growth and development. How plants perceive the external signals, then integrate and transduce the secondary signals specifically to the desired destination, is a fundamental characteristic of the MSP signaling network. The TCS elements involved in the MSP pathway and molecular mechanisms of signal transduction have been best understood in the model plant Arabidopsis thaliana. In this review, we focus on updated knowledge on TCS signal transduction in Arabidopsis. We first present a brief description of the TCS elements; then, the protein-protein interaction network is established. Finally, we discuss the possible molecular mechanisms involved in the specificity of the MSP signaling at the mRNA and protein levels.
Assuntos
Arabidopsis/fisiologia , Peptídeos e Proteínas de Sinalização Intracelular/fisiologia , Proteínas de Plantas/fisiologia , Mapas de Interação de Proteínas/fisiologia , Transdução de Sinais/fisiologia , Alquil e Aril Transferases/genética , Alquil e Aril Transferases/fisiologia , Arabidopsis/genética , Regulação da Expressão Gênica de Plantas , Histidina Quinase/genética , Histidina Quinase/fisiologia , Peptídeos e Proteínas de Sinalização Intracelular/genética , Magnésio/metabolismo , Modelos Biológicos , Família Multigênica , Fosfatos/metabolismo , Fosforilação , Fosfotransferases/genética , Fosfotransferases/fisiologia , Fitocromo/fisiologia , Proteínas de Plantas/genética , Ligação Proteica , Domínios Proteicos , Mapeamento de Interação de Proteínas , Processamento de Proteína Pós-Traducional , Proteólise , RNA Mensageiro/genética , RNA de Plantas/genética , Transdução de Sinais/genéticaRESUMO
Alkylation of guanine bases in DNA is detrimental to cells due to its high mutagenic and cytotoxic potential and is repaired by the alkyltransferase AGT. Additionally, alkyltransferase-like proteins (ATLs), which are structurally similar to AGTs, have been identified in many organisms. While ATLs are per se catalytically inactive, strong evidence has suggested that ATLs target alkyl lesions to the nucleotide excision repair system (NER). Using a combination of single-molecule and ensemble approaches, we show here recruitment of UvrA, the initiating enzyme of prokaryotic NER, to an alkyl lesion by ATL. We further characterize lesion recognition by ATL and directly visualize DNA lesion search by highly motile ATL and ATL-UvrA complexes on DNA at the molecular level. Based on the high similarity of ATLs and the DNA-interacting domain of AGTs, our results provide important insight in the lesion search mechanism, not only by ATL but also by AGT, thus opening opportunities for controlling the action of AGT for therapeutic benefit during chemotherapy.
Assuntos
Adenosina Trifosfatases/metabolismo , Alquil e Aril Transferases/metabolismo , Reparo do DNA/fisiologia , Proteínas de Ligação a DNA/metabolismo , Proteínas de Escherichia coli/metabolismo , Alquil e Aril Transferases/genética , Alquil e Aril Transferases/fisiologia , Alquilação/fisiologia , DNA/metabolismo , Dano ao DNA , Escherichia coli/metabolismo , Proteínas de Escherichia coli/fisiologia , Guanina/metabolismo , Microscopia de Força Atômica/métodos , Mutagênese , O(6)-Metilguanina-DNA Metiltransferase/genética , Pinças ÓpticasRESUMO
Mutations in mitochondrial DNA as well as nuclear-encoded mitochondrial proteins have been reported to cause tubulointerstitial kidney diseases and focal segmental glomerulosclerosis (FSGS). Recently, genes and pathways affecting mitochondrial turnover and permeability have been implicated in adult-onset FSGS. Furthermore, dysfunctioning mitochondria may be capable of engaging intracellular innate immune-sensing pathways. To determine the impact of mitochondrial dysfunction in FSGS and secondary innate immune responses, we generated Cre/loxP transgenic mice to generate a loss-of-function deletion mutation of the complex IV assembly cofactor heme A:farnesyltransferase (COX10) restricted to cells of the developing nephrons. These mice develop severe, early-onset FSGS with innate immune activation and die prematurely with kidney failure. Mutant kidneys showed loss of glomerular and tubular epithelial function, epithelial apoptosis, and, in addition, a marked interferon response. In vitro modeling of Cox10 deletion in primary kidney epithelium compromises oxygen consumption, ATP generation, and induces oxidative stress. In addition, loss of Cox10 triggers a selective interferon response, which may be caused by the leak of mitochondrial DNA into the cytosol activating the intracellular DNA sensor, stimulator of interferon genes. This new animal model provides a mechanism to study mitochondrial dysfunction in vivo and demonstrates a direct link between mitochondrial dysfunction and intracellular innate immune response.
Assuntos
Alquil e Aril Transferases/fisiologia , Modelos Animais de Doenças , Glomerulosclerose Segmentar e Focal/etiologia , Fatores Reguladores de Interferon/metabolismo , Interferons/farmacologia , Proteínas de Membrana/fisiologia , Estresse Oxidativo , Deleção de Sequência , Animais , Antivirais/farmacologia , Feminino , Glomerulosclerose Segmentar e Focal/metabolismo , Glomerulosclerose Segmentar e Focal/patologia , Fatores Reguladores de Interferon/genética , Masculino , Camundongos , Camundongos Transgênicos , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/imunologia , Mitocôndrias/patologiaRESUMO
Iron (Fe) is a vital trace element in plants, and deficiency of this element in apple trees can reduce fruit quality. Nicotianamine (NA) is known to play an important role in Fe transport and endogenous hormone balance. In the present study, we investigated the role of a nicotianamine synthase 1 gene (MxNas1) in an apple species, Malus xiaojinensis, that has a more Fe-efficient genotype than other apple species and ecotypes. To characterise the response of M. xiaojinensis to Fe deficiency, we used quantitative Q-PCR to determine the level of expression of MxNas1 and Western blot to measure protein levels. Immunohistochemical staining and GFP fluorescence localisation of the MxNAS1 protein were also carried out. HPLC and polarised absorption spectrophotometry were performed to investigate the effects of overexpression of MxNas1 in order to elucidate the role of MxNAS1 in the cellular uptake of active Fe in tobacco suspension cells. We found that MxNas1 expression and protein levels were higher under Fe deficiency stress than under Fe sufficiency. Immunohistochemical staining showed that MxNAS1 was localised mainly in the epidermal and vascular tissues of the roots, vascular tissues of the stem and palisade cells of mature leaves, and in parenchyma cells of young leaves. MxNAS1 was mainly localised in the plasma membranes and vesicles of protoplasts. In addition, overexpression of MxNas1 in stable transgenic tobacco cells increased NA and active Fe content under Fe sufficiency. The results suggest that MxNas1 expression in M. xiaojinensis is induced in response to Fe deficiency stress, resulting in higher levels of the protein. MxNAS1 may be involved in the redistribution of Fe in M. xiaojinensis under Fe deficiency.
Assuntos
Alquil e Aril Transferases/metabolismo , Deficiências de Ferro , Malus/fisiologia , Proteínas de Plantas/metabolismo , Alquil e Aril Transferases/fisiologia , Western Blotting , Regulação da Expressão Gênica de Plantas , Ferro/metabolismo , Malus/enzimologia , Malus/metabolismo , Reação em Cadeia da Polimerase em Tempo Real , Estresse FisiológicoRESUMO
KEY POINTS: Neurodegenerative disorders can exhibit dysfunctional mitochondrial respiratory chain complex IV activity. Conditional deletion of cytochrome c oxidase, the terminal enzyme in the respiratory electron transport chain of mitochondria, from hippocampal dentate granule cells in mice does not affect low-frequency dentate to CA3 glutamatergic synaptic transmission. High-frequency dentate to CA3 glutamatergic synaptic transmission and feedforward inhibition are significantly attenuated in cytochrome c oxidase-deficient mice. Intact presynaptic mitochondrial function is critical for the short-term dynamics of mossy fibre to CA3 synaptic function. ABSTRACT: Neurodegenerative disorders are characterized by peripheral and central symptoms including cognitive impairments which have been associated with reduced mitochondrial function, in particular mitochondrial respiratory chain complex IV or cytochrome c oxidase activity. In the present study we conditionally removed a key component of complex IV, protohaem IX farnesyltransferase encoded by the COX10 gene, in granule cells of the adult dentate gyrus. Utilizing whole-cell patch-clamp recordings from morphologically identified CA3 pyramidal cells from control and complex IV-deficient mice, we found that reduced mitochondrial function did not result in overt deficits in basal glutamatergic synaptic transmission at the mossy-fibre synapse because the amplitude, input-output relationship and 50 ms paired-pulse facilitation were unchanged following COX10 removal from dentate granule cells. However, trains of stimuli given at high frequency (> 20 Hz) resulted in dramatic reductions in short-term facilitation and, at the highest frequencies (> 50 Hz), also reduced paired-pulse facilitation, suggesting a requirement for adequate mitochondrial function to maintain glutamate release during physiologically relevant activity patterns. Interestingly, local inhibition was reduced, suggesting the effect observed was not restricted to synapses with CA3 pyramidal cells via large mossy-fibre boutons, but rather to all synapses formed by dentate granule cells. Therefore, presynaptic mitochondrial function is critical for the short-term dynamics of synapse function, which may contribute to the cognitive deficits observed in pathological mitochondrial dysfunction.
Assuntos
Alquil e Aril Transferases/fisiologia , Região CA3 Hipocampal/fisiologia , Giro Denteado/fisiologia , Proteínas de Membrana/fisiologia , Fibras Musgosas Hipocampais/fisiologia , Células Piramidais/fisiologia , Alquil e Aril Transferases/genética , Animais , Proteínas de Membrana/genética , Camundongos Transgênicos , Transmissão SinápticaRESUMO
Seed germination is an important stage in the lifecycle of a plant because it determines subsequent vegetative growth and reproduction. Here, we show that the E3 SUMO ligase AtSIZ1 regulates seed dormancy and germination. The germination rates of the siz1 mutants were less than 50%, even after a short period of ripening. However, their germination rates increased to wild-type levels after cold stratification or long periods of ripening. In addition, exogenous gibberellin (GA) application improved the germination rates of the siz1 mutants to the wild-type level. In transgenic plants, suppression of AtSIZ1 caused rapid post-translational decay of SLEEPY1 (SLY1), a positive regulator of GA signaling, during germination, and inducible AtSIZ1 overexpression led to increased SLY1 levels. In addition, overexpressing wild-type SLY1 in transgenic sly1 mutants increased their germination ratios to wild-type levels, whereas the germination ratio of transgenic sly1 mutants overexpressing mSLY1 was similar to that of sly1. The germination ratios of siz1 mutant seeds in immature developing siliques were much lower than those of the wild-type. Moreover, SLY1 and DELAY OF GERMINATION 1 (DOG1) transcript levels were reduced in the siz1 mutants, whereas the transcript levels of DELLA and ABSCISIC ACID INSENSITIVE 3 (ABI3) were higher than those of the wild-type. Taken together, these results indicate that the reduced germination of the siz1 mutants results from impaired GA signaling due to low SLY1 levels and activity, as well as hyperdormancy due to high levels of expression of dormancy-related genes including DOG1.
Assuntos
Proteínas de Arabidopsis/fisiologia , Arabidopsis/fisiologia , Germinação/fisiologia , Ligases/fisiologia , Alquil e Aril Transferases/fisiologia , Temperatura Baixa , Germinação/efeitos dos fármacos , Giberelinas/farmacologia , Mutação/fisiologia , Dormência de Plantas/efeitos dos fármacos , Dormência de Plantas/fisiologia , Plantas Geneticamente Modificadas/fisiologia , Transdução de Sinais/fisiologiaRESUMO
The chemically most complex modification in eukaryotic rRNA is the conserved hypermodified nucleotide N1-methyl-N3-aminocarboxypropyl-pseudouridine (m(1)acp(3)Ψ) located next to the P-site tRNA on the small subunit 18S rRNA. While S-adenosylmethionine was identified as the source of the aminocarboxypropyl (acp) group more than 40 years ago the enzyme catalyzing the acp transfer remained elusive. Here we identify the cytoplasmic ribosome biogenesis protein Tsr3 as the responsible enzyme in yeast and human cells. In functionally impaired Tsr3-mutants, a reduced level of acp modification directly correlates with increased 20S pre-rRNA accumulation. The crystal structure of archaeal Tsr3 homologs revealed the same fold as in SPOUT-class RNA-methyltransferases but a distinct SAM binding mode. This unique SAM binding mode explains why Tsr3 transfers the acp and not the methyl group of SAM to its substrate. Structurally, Tsr3 therefore represents a novel class of acp transferase enzymes.
Assuntos
Alquil e Aril Transferases/fisiologia , RNA Ribossômico 18S/biossíntese , Saccharomyces cerevisiae/enzimologia , Alquil e Aril Transferases/química , Domínio Catalítico , Cristalografia por Raios X , Células HCT116 , Humanos , Ligação de Hidrogênio , Sequências Repetidas Invertidas , Modelos Moleculares , Ligação Proteica , Processamento Pós-Transcricional do RNA , RNA Ribossômico 18S/química , S-Adenosilmetionina/químicaRESUMO
Cytokinins (CKs) are phytohormones regulating plant growth and development as well as response to the environment. In order to evaluate their function in heat stress (HS) responses, the effect of CK elevation was determined during three types of HS - targeted to shoots, targeted to roots and applied to the whole plant. The early (30min) and longer term (3h) responses were followed at the hormonal, transcriptomic and proteomic levels in Arabidopsis transformants with dexamethasone-inducible expression of the CK biosynthetic gene isopentenyltransferase (ipt) and the corresponding wild-type (Col-0). Combination of hormonal and phenotypic analyses showed transient up-regulation of the CK/abscisic acid ratio, which controls stomatal aperture, to be more pronounced in the transformant. HS responses of the root proteome and Rubisco-immunodepleted leaf proteome were followed using 2-D gel electrophoresis and MALDI-TOF/TOF. More than 100 HS-responsive proteins were detected, most of them being modulated by CK increase. Proteome and transcriptome analyses demonstrated that CKs have longer term positive effects on the stress-related proteins and transcripts, as well as on the photosynthesis-related ones. Transient accumulation of CKs and stimulation of their signal transduction in tissue(s) not exposed to HS indicate that they are involved in plant stress responses.
Assuntos
Alquil e Aril Transferases/fisiologia , Arabidopsis/fisiologia , Citocininas/fisiologia , Ácido Abscísico/fisiologia , Arabidopsis/efeitos dos fármacos , Arabidopsis/metabolismo , Dexametasona/farmacologia , Perfilação da Expressão Gênica , Regulação da Expressão Gênica de Plantas/fisiologia , Resposta ao Choque Térmico/fisiologia , Temperatura Alta , Reguladores de Crescimento de Plantas/fisiologia , Raízes de Plantas/metabolismo , Raízes de Plantas/fisiologia , Brotos de Planta/metabolismo , Brotos de Planta/fisiologia , Proteômica , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/fisiologia , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por MatrizRESUMO
Isoprene is a naturally produced hydrocarbon emitted into the atmosphere by green plants. It is also a constituent of synthetic rubber and a potential biofuel. Microbial production of isoprene can become a sustainable alternative to the prevailing chemical production of isoprene from petroleum. In this work, sequence homology searches were conducted to find novel isoprene synthases. Candidate sequences were functionally expressed in Escherichia coli and the desired enzymes were identified based on an isoprene production assay. The activity of three enzymes was shown for the first time: expression of the candidate genes from Ipomoea batatas, Mangifera indica, and Elaeocarpus photiniifolius resulted in isoprene formation. The Ipomoea batatas isoprene synthase produced the highest amounts of isoprene in all experiments, exceeding the isoprene levels obtained by the previously known Populus alba and Pueraria montana isoprene synthases that were studied in parallel as controls.
Assuntos
Alquil e Aril Transferases/isolamento & purificação , Escherichia coli/genética , Alquil e Aril Transferases/química , Alquil e Aril Transferases/fisiologia , Sequência de Aminoácidos , Butadienos , Genoma Bacteriano , Hemiterpenos/biossíntese , Dados de Sequência Molecular , Pentanos , Homologia de SequênciaRESUMO
BACKGROUND: Cytokinins (CKs) are involved in response to various environmental cues, including salinity. It has been previously reported that enhancing CK contents improved salt stress tolerance in tomato. However, the underlying mechanisms of CK metabolism and signaling under salt stress conditions remain to be deciphered. RESULTS: Two tomato isopentenyltransferases, SlIPT3 and SlIPT4, were characterized in tomato and Arabidopsis. Both proteins displayed isopentenyltransferase (IPT) activity in vitro, while their encoding genes exhibited different spatio-temporal expression patterns during tomato plant development. SlIPT3 and SlIPT4 were affected by the endogenous CK status, tightly connected with CKs feedback regulation, as revealed by hormonal treatements. In response to salt stress, SlIPT3 and SlIPT4 were strongly repressed in tomato roots, and differently affected in young and old leaves. SlIPT3 overexpression in tomato resulted in high accumulation of different CK metabolites, following modifications of CK biosynthesis-, signaling- and degradation-gene expression. In addition, 35S::SlIPT3 tomato plants displayed improved tolerance to salinity consecutive to photosynthetic pigments and K(+)/Na(+) ratio retention. Involvement of SlIPT3 and SlIPT4 in salt stress response was also observed in Arabidopsis ipt3 knock-out complemented plants, through maintenance of CK homeostasis. CONCLUSIONS: SlIPT3 and SlIPT4 are functional IPTs encoded by differently expressed genes, distinctively taking part in the salinity response. The substantial participation of SlIPT3 in CK metabolism during salt stress has been determined in 35S::SlIPT3 tomato transformants, where enhancement of CKs accumulation significantly improved plant tolerance to salinity, underlining the importance of this phytohormone in stress response.
Assuntos
Alquil e Aril Transferases/fisiologia , Arabidopsis/fisiologia , Citocininas/metabolismo , Regulação da Expressão Gênica de Plantas , Tolerância ao Sal , Solanum lycopersicum/enzimologia , Solanum lycopersicum/fisiologia , Alquil e Aril Transferases/genética , Arabidopsis/genética , Solanum lycopersicum/embriologia , Solanum lycopersicum/genética , Plantas Geneticamente Modificadas/genética , Plantas Geneticamente Modificadas/fisiologiaRESUMO
Chrysanthemyl diphosphate synthase (CDS) is the first pathway-specific enzyme in the biosynthesis of pyrethrins, the most widely used plant-derived pesticide. CDS catalyzes c1'-2-3 cyclopropanation reactions of two molecules of dimethylallyl diphosphate (DMAPP) to yield chrysanthemyl diphosphate (CPP). Three proteins are known to catalyze this cyclopropanation reaction of terpene precursors. Two of them, phytoene and squalene synthase, are bifunctional enzymes with both prenyltransferase and terpene synthase activity. CDS, the other member, has been reported to perform only the prenyltransferase step. Here we show that the NDXXD catalytic motif of CDS, under the lower substrate conditions prevalent in plants, also catalyzes the next step, converting CPP into chrysanthemol by hydrolyzing the diphosphate moiety. The enzymatic hydrolysis reaction followed conventional Michaelis-Menten kinetics, with a Km value for CPP of 196 µm. For the chrysanthemol synthase activity, DMAPP competed with CPP as substrate. The DMAPP concentration required for half-maximal activity to produce chrysanthemol was â¼100 µm, and significant substrate inhibition was observed at elevated DMAPP concentrations. The N-terminal peptide of CDS was identified as a plastid-targeting peptide. Transgenic tobacco plants overexpressing CDS emitted chrysanthemol at a rate of 0.12-0.16 µg h(-1) g(-1) fresh weight. We propose that CDS should be renamed a chrysanthemol synthase utilizing DMAPP as substrate.
Assuntos
Alquil e Aril Transferases/fisiologia , Chrysanthemum cinerariifolium/enzimologia , Proteínas de Plantas/fisiologia , Terpenos/metabolismo , Alquil e Aril Transferases/genética , Sequência de Aminoácidos , Vias Biossintéticas , Dados de Sequência Molecular , Proteínas de Plantas/genética , Plantas Geneticamente Modificadas/genética , Nicotiana/genéticaRESUMO
Synaptic impairment rather than neuronal loss may be the leading cause of cognitive dysfunction in brain aging. Certain small Rho-GTPases are involved in synaptic plasticity, and their dysfunction is associated with brain aging and neurodegeneration. Rho-GTPases undergo prenylation by attachment of geranylgeranylpyrophosphate (GGPP) catalyzed by GGTase-I. We examined age-related changes in the abundance of Rho and Rab proteins in membrane and cytosolic fractions as well as of GGTase-I in brain tissue of 3- and 23-month-old C57BL/6 mice. We report a shift in the cellular localization of Rho-GTPases toward reduced levels of membrane-associated and enhanced cytosolic levels of those proteins in aged mouse brain as compared with younger mice. The age-related reduction in membrane-associated Rho proteins was associated with a reduction in GGTase-Iß levels that regulates binding of GGPP to Rho-GTPases. Proteins prenylated by GGTase-II were not reduced in aged brain indicating a specific targeting of GGTase-I in the aged brain. Inhibition of GGTase-I in vitro modeled the effects of aging we observed in vivo. We demonstrate for the first time a decrease in membrane-associated Rho proteins in aged brain in association with down-regulation of GGTase-Iß. This down-regulation could be one of the mechanisms causing age-related weakening of synaptic plasticity.
Assuntos
Envelhecimento/metabolismo , Alquil e Aril Transferases/fisiologia , Cérebro/metabolismo , Proteínas de Membrana/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Prenilação de Proteína , Membranas Sinápticas/metabolismo , Proteínas rho de Ligação ao GTP/metabolismo , Envelhecimento/psicologia , Alquil e Aril Transferases/antagonistas & inibidores , Animais , Linhagem Celular Tumoral , Transtornos Cognitivos/etiologia , Regulação Enzimológica da Expressão Gênica , Humanos , Imidazóis/farmacologia , Leucina/análogos & derivados , Leucina/farmacologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Naftalenos/farmacologia , Neuroblastoma/patologia , RNA Mensageiro/biossíntese , Transmissão Sináptica , Terpenos/metabolismo , Proteína cdc42 de Ligação ao GTP/metabolismo , Proteínas rab de Ligação ao GTP/metabolismo , Proteínas rac1 de Ligação ao GTP/metabolismo , Proteína rhoA de Ligação ao GTP/metabolismoRESUMO
Leukocyte infiltration and acinar cell necrosis are hallmarks of severe AP, but the signaling pathways regulating inflammation and organ injury in the pancreas remain elusive. In the present study, we investigated the role of geranylgeranyltransferase in AP. Male C57BL/6 mice were treated with a geranylgeranyltransferase inhibitor GGTI-2133 (20 mg/kg) prior to induction of pancreatitis by infusion of taurocholate into the pancreatic duct. Pretreatment with GGTI-2133 reduced plasma amylase levels, pancreatic neutrophil recruitment, hemorrhage, and edema formation in taurocholate-evoked pancreatitis. Moreover, administration of GGTI-2133 decreased the taurocholate-induced increase of MPO activity in the pancreas and lung. Treatment with GGTI-2133 markedly reduced levels of CXCL2 in the pancreas and IL-6 in the plasma in response to taurocholate challenge. Notably, geranylgeranyltransferase inhibition abolished neutrophil expression of Mac-1 in mice with pancreatitis. Finally, inhibition of geranylgeranyltransferase had no direct effect on secretagogue-induced activation of trypsinogen in pancreatic acinar cells in vitro. A significant role of geranylgeranyltransferase was confirmed in an alternate model of AP induced by L-arginine challenge. Our findings show that geranylgeranyltransferase regulates neutrophil accumulation and tissue damage via expression of Mac-1 on neutrophils and CXCL2 formation in AP. Thus, these results reveal new signaling mechanisms in pancreatitis and indicate that targeting geranylgeranyltransferase might be an effective way to ameliorate severe AP.
Assuntos
Alquil e Aril Transferases/fisiologia , Imidazóis/farmacologia , Leucina/análogos & derivados , Naftalenos/farmacologia , Neutrófilos/fisiologia , Pancreatite/imunologia , Células Acinares/enzimologia , Doença Aguda , Alquil e Aril Transferases/antagonistas & inibidores , Animais , Quimiocina CXCL2/fisiologia , Leucina/farmacologia , Antígeno de Macrófago 1/fisiologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Infiltração de Neutrófilos , Pancreatite/tratamento farmacológico , Prenilação , Tripsinogênio/metabolismo , Proteínas rac1 de Ligação ao GTP/metabolismoRESUMO
The multidrug resistance 1 (MDR1) gene product P-glycoprotein is an ATP-dependent efflux pump associated with chemotherapy failure in breast cancer. In the present study, we show that paclitaxel induces MDR1 expression in the MCF-7 breast cancer cell line in a MAPK/Egr-1-dependent manner. Paclitaxel exposure activated the Erk1/2/MAPK pathway and promoted the accumulation of the early response transcription factor Egr-1 in MCF-7 cells. Egr-1 binds to the GC element on the proximal MDR1 promoter to enhance MDR1 transcription. Loss of Egr-1 function in paclitaxel-resistant MCF-7 cells decreased MDR1 expression, whereas inhibiting Erk1/2 activity reduced both Egr-1 accumulation and MDR1 expression. These findings suggest that Erk1/2-induced Egr-1 accumulation activates MDR1 transcription and thereby induces the drug resistance observed in paclitaxel-resistant MCF-7 cells. Further mechanistic studies indicate that Egr-1 most likely does not induce the constitutive activation of Erk1/2 through its target gene geranylgeranyl diphosphate synthase (GGPPS), which regulates Ras prenylation. Indeed, our results suggest a novel pathway by which paclitaxel induces MDR1 expression, possibly illuminating a potential target pathway for the prevention of MDR1-mediated drug resistance.
Assuntos
Membro 1 da Subfamília B de Cassetes de Ligação de ATP/biossíntese , Alquil e Aril Transferases/fisiologia , Neoplasias da Mama/metabolismo , Dimetilaliltranstransferase/fisiologia , Resistencia a Medicamentos Antineoplásicos/fisiologia , Proteína 1 de Resposta de Crescimento Precoce/fisiologia , Farnesiltranstransferase/fisiologia , Regulação Neoplásica da Expressão Gênica , Geraniltranstransferase/fisiologia , Subfamília B de Transportador de Cassetes de Ligação de ATP , Membro 1 da Subfamília B de Cassetes de Ligação de ATP/genética , Antineoplásicos Fitogênicos/farmacologia , Antineoplásicos Fitogênicos/uso terapêutico , Sítios de Ligação/fisiologia , Neoplasias da Mama/terapia , Proteína 1 de Resposta de Crescimento Precoce/metabolismo , Feminino , Humanos , Células MCF-7 , Paclitaxel/farmacologia , Paclitaxel/uso terapêuticoRESUMO
Geranylgeranyltransferase I (GGT) is a prenyltransferase that mediates lipid modification of Rho small GTPases, such as Rho, Rac, and Cdc42, which are important for neuronal synaptogenesis. Although GGT is expressed in brain extensively, the function of GGT in central nerves system is largely unknown so far. We have previously demonstrated that GGT promotes the basal and neuronal activity and brain-derived neurotrophic factor (BDNF)-induced dendritic morphogenesis of cultured hippocampal neurons and cerebellar slices. This study is to explore the function and mechanism of GGT in neuronal synaptogenesis. We found that the protein level and activity of GGT gradually increased in rat hippocampus from P7 to P28 and subcellular located at synapse of neurons. The linear density of Synapsin 1 and post-synaptic density protein 95 increased by over-expression of GGT ß, while reduced by inhibition or down-regulation of GGT. In addition, GGT and its known substrate Rac was activated by BDNF, which promotes synaptogenesis in cultured hippocampal neurons. Furthermore, BDNF-induced synaptogenesis was eliminated by GGT inhibition or down-regulation, as well as by non-prenylated Rac1 over-expression. Together, our data suggested that GGT mediates BDNF-induced neuronal synaptogenesis through Rac1 activation.
Assuntos
Alquil e Aril Transferases/fisiologia , Fator Neurotrófico Derivado do Encéfalo/farmacologia , Neurogênese/fisiologia , Sinapses/enzimologia , Animais , Fator Neurotrófico Derivado do Encéfalo/fisiologia , Células Cultivadas , Hipocampo/efeitos dos fármacos , Hipocampo/enzimologia , Humanos , Neurogênese/efeitos dos fármacos , Ratos , Ratos Sprague-Dawley , Sinapses/efeitos dos fármacos , Proteínas rac1 de Ligação ao GTP/fisiologiaRESUMO
Streptococcal toxic shock syndrome is most frequently associated with Streptococcus pyogenes of the M1 serotype. Simvastatin protects against M1 protein-induced acute lung damage, although downstream mechanisms remain elusive. Herein, we hypothesized that geranylgeranylation might regulate proinflammatory effects in M1 protein-induced lung injury. Male C57BL/6 mice received the geranylgeranyl transferase inhibitor, GGTI-2133, before M1 protein injection. Bronchoalveolar fluid and lung tissue were harvested for quantification of neutrophil recruitment, edema, and CXC chemokine formation. Mac-1 expression on neutrophils was quantified by use of flow cytometry. Quantitative reverse transcriptase-polymerase chain reaction was used to determine gene expression of CXC chemokines in alveolar macrophages. GGTI-2133 reduced M1 protein-provoked infiltration of neutrophils, edema, and tissue injury in the lung. Inhibition of geranylgeranyl transferase had no effect on M1 protein-evoked upregulation of Mac-1 on neutrophils. However, geranylgeranyl transferase inhibition completely inhibited pulmonary formation of CXC chemokines in mice exposed to M1 protein. Notably, GGTI-2133 abolished M1 protein-induced gene expression of CXC chemokines in alveolar macrophages. These novel findings indicate that geranylgeranyl transferase is an important regulator of neutrophil recruitment and CXC chemokine production in the lung. Thus, targeting geranylgeranyl transferase might be a potent way to ameliorate streptococcal M1 protein-triggered acute lung injury.
Assuntos
Lesão Pulmonar Aguda/enzimologia , Alquil e Aril Transferases/fisiologia , Quimiocinas CXC/biossíntese , Lesão Pulmonar Aguda/induzido quimicamente , Lesão Pulmonar Aguda/patologia , Lesão Pulmonar Aguda/prevenção & controle , Alquil e Aril Transferases/antagonistas & inibidores , Animais , Antígenos de Bactérias , Proteínas da Membrana Bacteriana Externa , Proteínas de Transporte , Quimiotaxia de Leucócito/efeitos dos fármacos , Inibidores Enzimáticos/uso terapêutico , Imidazóis/uso terapêutico , Leucina/análogos & derivados , Leucina/uso terapêutico , Pulmão/metabolismo , Antígeno de Macrófago 1/biossíntese , Macrófagos Alveolares/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Naftalenos/uso terapêutico , Infiltração de Neutrófilos/efeitos dos fármacos , Infiltração de Neutrófilos/fisiologia , Neutrófilos/metabolismo , Regulação para Cima/efeitos dos fármacosRESUMO
Endogenous and exogenous factors constantly challenge cellular DNA, generating cytotoxic and/or mutagenic DNA adducts. As a result, organisms have evolved different mechanisms to defend against the deleterious effects of DNA damage. Among these diverse repair pathways, direct DNA-repair systems provide cells with simple yet efficient solutions to reverse covalent DNA adducts. In this review, we focus on recent advances in the field of direct DNA repair, namely, photolyase-, alkyltransferase-, and dioxygenase-mediated repair processes. We present specific examples to describe new findings of known enzymes and appealing discoveries of new proteins. At the end of this article, we also briefly discuss the influence of direct DNA repair on other fields of biology and its implication on the discovery of new biology.