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1.
PLoS One ; 15(3): e0230572, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32210477

RESUMO

Chromatin structure plays a decisive role in gene regulation through the actions of transcriptional activators, coactivators, and epigenetic machinery. These trans-acting factors contribute to gene expression through their interactions with chromatin structure. In yeast INO1 activation, transcriptional activators and coactivators have been defined through intense study but the mechanistic links within these trans-acting factors and their functional implications are not yet fully understood. In this study, we examined the crosstalk within transcriptional coactivators with regard to the implications of Snf2p acetylation during INO1 activation. Through various biochemical analysis, we demonstrated that both Snf2p and Ino80p chromatin remodelers accumulate at the INO1 promoter in the absence of Snf2p acetylation during induction. Furthermore, nucleosome density and histone acetylation patterns remained unaffected by Snf2p acetylation status. We also showed that cells experience increased sensitivity to copper toxicity when remodelers accumulate at the INO1 promoter due to the decreased CUP1 expression. Therefore, our data provide evidence for crosstalk within transcriptional co-activators during INO1 activation. In light of these findings, we propose a model in which acetylation-driven chromatin remodeler recycling allows for efficient regulation of genes that are dependent upon limited co-activators.


Assuntos
Adenosina Trifosfatases/metabolismo , Metalotioneína/metabolismo , Mio-Inositol-1-Fosfato Sintase/genética , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Fatores de Transcrição/metabolismo , Acetilação , Adenosina Trifosfatases/genética , Sobrevivência Celular/efeitos dos fármacos , Cromatina/metabolismo , Montagem e Desmontagem da Cromatina , Cobre/metabolismo , Cobre/toxicidade , Histonas/metabolismo , Metalotioneína/genética , Mio-Inositol-1-Fosfato Sintase/metabolismo , Nucleossomos/metabolismo , Regiões Promotoras Genéticas , Fatores de Transcrição/genética , Ativação Transcricional
2.
Dev Cell ; 52(3): 309-320.e5, 2020 02 10.
Artigo em Inglês | MEDLINE | ID: mdl-31902656

RESUMO

Movement of chromosome sites within interphase cells is critical for numerous pathways including RNA transcription and genome organization. Yet, a mechanism for reorganizing chromatin in response to these events had not been reported. Here, we delineate a molecular chaperone-dependent pathway for relocating activated gene loci in yeast. Our presented data support a model in which a two-authentication system mobilizes a gene promoter through a dynamic network of polymeric nuclear actin. Transcription factor-dependent nucleation of a myosin motor propels the gene locus through the actin matrix, and fidelity of the actin association was ensured by ARP-containing chromatin remodelers. Motor activity of nuclear myosin was dependent on the Hsp90 chaperone. Hsp90 further contributed by biasing the remodeler-actin interaction toward nucleosomes with the non-canonical histone H2A.Z, thereby focusing the pathway on select sites such as transcriptionally active genes. Together, the system provides a rapid and effective means to broadly yet selectively mobilize chromatin sites.


Assuntos
Montagem e Desmontagem da Cromatina , Cromossomos Fúngicos , Regulação Fúngica da Expressão Gênica , Histonas/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Ativação Transcricional , Actinas/genética , Actinas/metabolismo , Adenosina Trifosfatases/genética , Adenosina Trifosfatases/metabolismo , Núcleo Celular/genética , Núcleo Celular/metabolismo , Proteínas do Citoesqueleto/genética , Proteínas do Citoesqueleto/metabolismo , Proteínas de Choque Térmico HSP90/genética , Proteínas de Choque Térmico HSP90/metabolismo , Histonas/genética , Mio-Inositol-1-Fosfato Sintase/genética , Mio-Inositol-1-Fosfato Sintase/metabolismo , Cadeias Pesadas de Miosina/genética , Cadeias Pesadas de Miosina/metabolismo , Nucleossomos/genética , Nucleossomos/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/crescimento & desenvolvimento , Proteínas de Saccharomyces cerevisiae/genética
3.
Biochemistry ; 58(51): 5112-5116, 2019 12 24.
Artigo em Inglês | MEDLINE | ID: mdl-31825604

RESUMO

The myo-inositol-1-phosphate synthase (MIPS) ortholog Ari2, which is encoded in the aristeromycin biosynthetic gene cluster, catalyzes the formation of five-membered cyclitol phosphate using d-fructose 6-phosphate (F6P) as a substrate. To understand the stereochemistry during the Ari2 reaction in vivo, we carried out feeding experiments with (6S)-d-[6-2H1]- and (6R)-d-[6-2H1]glucose in the aristeromycin-producing strain Streptomyces citricolor. We observed retention of the 2H atom of (6S)-d-[6-2H1]glucose and no incorporation of the 2H atom from (6R)-d-[6-2H1]glucose in aristeromycin. This indicates that Ari2 abstracts the pro-R proton at C6 of F6P after oxidation of C5-OH by nicotinamide adenine dinucleotide (NAD+) to generate the enolate intermediate, which then attacks the C2 ketone to form the C-C bond via aldol-type condensation. The reaction of Ari2 with (6S)-d-[6-2H1]- and (6R)-d-[6-2H1]F6P in vitro exhibited identical stereochemistry compared with that observed during the feeding experiments. Furthermore, analysis of the crystal structure of Ari2, including NAD+ as a ligand, revealed the active site of Ari2 to be similar to that of MIPS of Mycobacterium tuberculosis, supporting the similarity of the reaction mechanisms of Ari2 and MIPS.


Assuntos
Adenosina/análogos & derivados , Mio-Inositol-1-Fosfato Sintase/metabolismo , Adenosina/biossíntese , Adenosina/química , Modelos Moleculares , Mio-Inositol-1-Fosfato Sintase/química , Conformação Proteica , Estereoisomerismo , Streptomyces/enzimologia
4.
Sci Rep ; 9(1): 7744, 2019 05 23.
Artigo em Inglês | MEDLINE | ID: mdl-31123331

RESUMO

Phytic acid (PA), the major phosphorus reserve in soybean seeds (60-80%), is a potent ion chelator, causing deficiencies that leads to malnutrition. Several forward and reverse genetics approaches have ever since been explored to reduce its phytate levels to improve the micronutrient and phosphorous availability. Transgenic technology has met with success by suppressing the expression of the PA biosynthesis-related genes in several crops for manipulating their phytate content. In our study, we targeted the disruption of the expression of myo-inositol-3-phosphate synthase (MIPS1), the first and the rate limiting enzyme in PA biosynthesis in soybean seeds, by both antisense (AS) and RNAi approaches, using a seed specific promoter, vicilin. PCR and Southern analysis revealed stable integration of transgene in the advanced progenies. The transgenic seeds (T4) of AS (MS14-28-12-29-3-5) and RNAi (MI51-32-22-1-13-6) soybean lines showed 38.75% and 41.34% reduction in phytate levels respectively, compared to non-transgenic (NT) controls without compromised growth and seed development. The electron microscopic examination also revealed reduced globoid crystals in the Protein storage vacoules (PSVs) of mature T4 seeds compared to NT seed controls. A significant increase in the contents of Fe2+ (15.4%, 21.7%), Zn2+ (7.45%, 11.15%) and Ca2+ (10.4%, 15.35%) were observed in MS14-28-12-29-3-5 and MI51-32-22-1-13-6 transgenic lines, respectively, compared to NT implicating improved mineral bioavailability. This study signifies proof-of-concept demonstration of seed-specific PA reduction and paves the path towards low phytate soybean through pathway engineering using the new and precise editing tools.


Assuntos
Mio-Inositol-1-Fosfato Sintase/genética , Ácido Fítico/metabolismo , Soja/genética , Disponibilidade Biológica , Fabaceae/genética , Fabaceae/crescimento & desenvolvimento , Regulação da Expressão Gênica de Plantas/genética , Engenharia Genética/métodos , Minerais/metabolismo , Mio-Inositol-1-Fosfato Sintase/metabolismo , Fósforo/metabolismo , Ácido Fítico/efeitos adversos , Ácido Fítico/química , Plantas Geneticamente Modificadas/genética , Plantas Geneticamente Modificadas/metabolismo , Regiões Promotoras Genéticas/genética , Interferência de RNA/fisiologia , RNA Antissenso/genética , Proteínas de Armazenamento de Sementes/genética , Sementes/genética , Soja/crescimento & desenvolvimento
5.
Int J Mol Sci ; 20(10)2019 May 15.
Artigo em Inglês | MEDLINE | ID: mdl-31096655

RESUMO

d-pinitol is the most commonly accumulated sugar alcohol in the Leguminosae family and has been observed to increase significantly in response to abiotic stress. While previous studies have identified genes involved in d-pinitol synthesis, no study has investigated transcript expression in planta. The present study quantified the expression of several genes involved in d-pinitol synthesis in different plant tissues and investigated the accumulation of d-pinitol, myo-inositol and other metabolites in response to a progressive soil drought in soybean (Glycine max). Expression of myo-inositol 1-phosphate synthase (INPS), the gene responsible for the conversion of glucose-6-phosphate to myo-inositol-1-phosphate, was significantly up regulated in response to a water deficit for the first two sampling weeks. Expression of myo-inositol O-methyl transferase (IMT1), the gene responsible for the conversion of myo-inositol into d-ononitol was only up regulated in stems at sampling week 3. Assessment of metabolites showed significant changes in their concentration in leaves and stems. d-Pinitol concentration was significantly higher in all organs sampled from water deficit plants for all three sampling weeks. In contrast, myo-inositol, had significantly lower concentrations in leaf samples despite up regulation of INPS suggesting the transcriptionally regulated flux of carbon through the myo-inositol pool is important during water deficit.


Assuntos
Inositol/análogos & derivados , Soja/genética , Soja/metabolismo , Água/metabolismo , Secas , Regulação Enzimológica da Expressão Gênica , Regulação da Expressão Gênica de Plantas , Genes de Plantas/genética , Glucose/metabolismo , Glucose-6-Fosfato/metabolismo , Inositol/biossíntese , Inositol/genética , Metiltransferases/genética , Metiltransferases/metabolismo , Mio-Inositol-1-Fosfato Sintase/genética , Mio-Inositol-1-Fosfato Sintase/metabolismo , Folhas de Planta/metabolismo , Proteínas de Plantas/genética , Estresse Fisiológico , Sacarose/metabolismo , Transcriptoma
6.
J Agric Food Chem ; 67(17): 5043-5052, 2019 May 01.
Artigo em Inglês | MEDLINE | ID: mdl-30977368

RESUMO

The low phytic acid ( lpa) soybean ( Glycine max L. Merr.) mutant Gm-lpa-TW-1-M, resulting from a 2 bp deletion in GmMIPS1, was crossed with a commercial cultivar. F3 and F5 progenies were subjected to nontargeted GC-based metabolite profiling, allowing analysis of a broad array of low molecular weight constituents. In the homozygous lpa mutant progenies the intended phytic acid reduction was accompanied by remarkable metabolic changes of nutritionally relevant constituents such as reduced contents of raffinose oligosaccharides and galactosyl cyclitols as well as increased concentrations in sucrose and various free amino acids. The mutation-induced metabolite signature was nearly unaffected by the cross-breeding and consistently expressed over generations and in different growing seasons. Therefore, not only the primary MIPS1 lpa mutant but also its progenies might be valuable genetic resources for commercial breeding programs to produce soybean seeds stably exhibiting improved phytate-related and nutritional properties.


Assuntos
Proteínas de Arabidopsis/genética , Mio-Inositol-1-Fosfato Sintase/genética , Ácido Fítico/análise , Proteínas de Plantas/genética , Soja/enzimologia , Proteínas de Arabidopsis/metabolismo , Homozigoto , Hibridização Genética , Mutação , Mio-Inositol-1-Fosfato Sintase/metabolismo , Oligossacarídeos/análise , Oligossacarídeos/metabolismo , Ácido Fítico/metabolismo , Melhoramento Vegetal , Proteínas de Plantas/metabolismo , Rafinose/análise , Rafinose/metabolismo , Soja/química , Soja/genética , Soja/metabolismo , Sacarose/análise , Sacarose/metabolismo
7.
Int J Mol Sci ; 20(5)2019 Mar 11.
Artigo em Inglês | MEDLINE | ID: mdl-30862084

RESUMO

Myo-inositol-1-phosphate synthase (MIPS, EC 5.5.1.4) plays important roles in plant growth and development, stress responses, and cellular signal transduction. MIPS genes were found preferably expressed during fiber cell initiation and early fast elongation in upland cotton (Gossypium hirsutum), however, current understanding of the function and regulatory mechanism of MIPS genes to involve in cotton fiber cell growth is limited. Here, by genome-wide analysis, we identified four GhMIPS genes anchoring onto four chromosomes in G. hirsutum and analyzed their phylogenetic relationship, evolutionary dynamics, gene structure and motif distribution, which indicates that MIPS genes are highly conserved from prokaryotes to green plants, with further exon-intron structure analysis showing more diverse in Brassicales plants. Of the four GhMIPS members, based on the significant accumulated expression of GhMIPS1D at the early stage of fiber fast elongating development, thereby, the GhMIPS1D was selected to investigate the function of participating in plant development and cell growth, with ectopic expression in the loss-of-function Arabidopsis mips1 mutants. The results showed that GhMIPS1D is a functional gene to fully compensate the abnormal phenotypes of the deformed cotyledon, dwarfed plants, increased inflorescence branches, and reduced primary root lengths in Arabidopsis mips1 mutants. Furthermore, shortened root cells were recovered and normal root cells were significantly promoted by ectopic expression of GhMIPS1D in Arabidopsis mips1 mutant and wild-type plants respectively. These results serve as a foundation for understanding the MIPS family genes in cotton, and suggest that GhMIPS1D may function as a positive regulator for plant cell elongation.


Assuntos
Arabidopsis/crescimento & desenvolvimento , Arabidopsis/genética , Genes de Plantas , Gossypium/genética , Mio-Inositol-1-Fosfato Sintase/genética , Raízes de Plantas/crescimento & desenvolvimento , Raízes de Plantas/genética , Motivos de Aminoácidos , Sequência de Aminoácidos , Sequência Conservada , Expressão Ectópica do Gene , Éxons , Regulação da Expressão Gênica de Plantas , Íntrons , Mutação com Perda de Função , Família Multigênica , Mio-Inositol-1-Fosfato Sintase/química , Mio-Inositol-1-Fosfato Sintase/metabolismo , Fenótipo , Filogenia
8.
Genomics ; 111(6): 1929-1945, 2019 12.
Artigo em Inglês | MEDLINE | ID: mdl-30660787

RESUMO

The myo-inositol biosynthesis pathway triggering protein MIPS is best known for its necessity, ubiquitous nature and occurrence throughout all living kingdom. However, the functional disparity of MIPS genes in green plant is still viable. The present work considered a comprehensive genome-wide analysis from sequenced plants to identify MIPS homologs in respective organisms and their genomic architecture. Variation of MIPS gene expression in twelve different species in diverse conditions has also been analysed. All MIPS genes share a conserved sequence property in most of its coding region, but its regulatory elements, gene structure and expression network vary significantly. Phylogenetic inference confirms the evolution of MIPS from a single common algal ancestor to seed plants and acquiring functional variation through genomic control. This paper represents MIPS as a model for studying gene duplication, functional divergence and diversification events in plant lineages.


Assuntos
Filogenia , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Plantas/genética , Arabidopsis/citologia , Arabidopsis/genética , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Sequência Conservada , Evolução Molecular , Duplicação Gênica , Regulação da Expressão Gênica de Plantas , Genoma de Planta , Inositol/metabolismo , Família Multigênica , Mio-Inositol-1-Fosfato Sintase/genética , Mio-Inositol-1-Fosfato Sintase/metabolismo , Plantas/metabolismo , Sequências Reguladoras de Ácido Nucleico
9.
Microbiologyopen ; 8(5): e00721, 2019 05.
Artigo em Inglês | MEDLINE | ID: mdl-30270521

RESUMO

Reactive oxygen species (ROS) generated in aerobic metabolism and oxidative stress lead to macromolecules damage, such as to proteins, lipids, and DNA, which can be eliminated by the redox buffer mycothiol (AcCys-GlcN-Ins, MSH). Myo-inositol-phosphate synthase (Ino-1) catalyzes the first committed step in the synthesis of MSH, thus playing a critical role in the growth of the organism. Although Ino-1s have been systematically studied in eukaryotes, their physiological and biochemical functions remain largely unknown in bacteria. In this study, we report that Ino-1 plays an important role in oxidative stress resistance in the gram-positive Actinobacteria Corynebacterium glutamicum. Deletion of the ino-1 gene resulted in a decrease in cell viability, an increase in ROS production, and the aggravation of protein carbonylation levels under various stress conditions. The physiological roles of Ino-1 in the resistance to oxidative stresses were corroborated by the absence of MSH in the Δino-1 mutant. In addition, we found that the homologous expression of Ino-1 in C. glutamicum yielded a functionally active protein, while when expressed in Escherichia coliBL21(DE3), it lacked measurable activity. An examination of the molecular mass (Mr) suggested that Ino-1 expressed in E. coliBL21(DE3) was not folded in a catalytically competent conformation. Together, the results unequivocally showed that Ino-1 was important for the mediation of oxidative resistance by C. glutamicum.


Assuntos
Corynebacterium glutamicum/enzimologia , Corynebacterium glutamicum/fisiologia , Cisteína/metabolismo , Glicopeptídeos/metabolismo , Inositol/metabolismo , Mio-Inositol-1-Fosfato Sintase/metabolismo , Estresse Oxidativo , Espécies Reativas de Oxigênio/metabolismo , Aerobiose , Deleção de Genes , Viabilidade Microbiana , Mio-Inositol-1-Fosfato Sintase/genética , Carbonilação Proteica
10.
J UOEH ; 40(3): 217-224, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30224617

RESUMO

Novel drugs possessing a mechanism of action specific to pathogenic mycobacteria, including Mycobacterium tuberculosis, are needed. In 2010, we discovered that the biosynthetic pathway of phosphatidylinositol, which is a membrane phospholipid, differs between humans and mycobacteria. The key enzyme responsible for this difference is phosphatidylinositol phosphate (PIP) synthase, which is present only in a few bacteria belonging to the phylum Actinobacteria. Discovering compounds that inhibit the activity of this enzyme will lead to the development of new drugs specific to pathogenic mycobacteria. Measuring PIP synthase activity requires the isotope-labeled substrate 1l-myo-inositol 1-phosphate (1l-Ino-1P). Because this substrate is not commercially available, we synthesized it from [14C] glucose 6-phosphate ([14C] Glc-6P), using a crude enzyme solution isolated from the methanoarchaeon 1l-Ino-1P synthase. The activity of 1l-Ino-1P synthase in the crude enzyme mixture was low, and quantitative analysis of the synthesized 1l-Ino-1P was inaccurate due to impurities present in the crude enzyme mixture. In the present study, we describe a method for synthesizing 1l-Ino-1P using a solution containing recombinant 1l-Ino-1P synthase derived from the hyperthermophilic archaeon Aeropyrum pernix. In addition, we elucidate the conditions leading to the almost complete conversion of Glc-6P into 1l-Ino-1P using this enzyme. Quantitation of the synthesized 1l -Ino-1P was performed by colorimetry and gas liquid chromatography. Further, we confirmed that isotope-labeled 1l-Ino-1P, which is difficult to quantitate by gas liquid chromatography, can be accurately quantified by colorimetry. We also confirmed that 1d-inositol 1-phosphate cannot be a substrate for PIP synthase.


Assuntos
Fosfatos de Inositol/metabolismo , Mycobacterium/enzimologia , Mio-Inositol-1-Fosfato Sintase/metabolismo , Colorimetria , Mio-Inositol-1-Fosfato Sintase/química , Especificidade por Substrato
11.
Planta ; 248(5): 1121-1141, 2018 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-30066217

RESUMO

MAIN CONCLUSION: The promoter deletion mutants from second isoform of INO1 (gene-encoding MIPS) from Porteresia coarctata of 932 bp (pPcINO1.2.932) and 793 bp (pPcINO1.2.793) prove to be very efficient as salt/drought stress-inducible promoters, while pPcINO1.2.932 is found to be responsive to cold stress as well. The promoters of the two identified myo-inositol-1-phosphate synthase (INO1) isoforms from salt-tolerant wild rice, Porteresia coarctata (PcINO1.1 and PcINO1.2) have been compared bioinformatically with their counterparts present in the salt-sensitive rice, Oryza sativa. PcINO1.2 promoter was found to be enriched with many abiotic stress-responsive elements, like abscisic acid-responsive elements, MYC-responsive elements, MYB-binding sites, low-temperature stress-responsive elements, and heat-shock elements similar to the ones found in the conserved motifs of the promoters of salt/drought stress-inducible INO1 promoters across Kingdom Planta. To have detailed analysis on the arrangement of cis-acting regulatory elements present in PcINO1 promoters, 5' deletion mutational studies were performed in dicot model plants. Both transient as well as stable transformation methods were used to check the influence of PcINO1 promoter deletion mutants under salt and physiologically drought conditions using ß-glucuronidase as the reporter gene. The deletion mutant from the promoter of PcINO1.2 of length 932 bp (pPcINO1.2.932) was found to be significantly upregulated under drought stress and also in cold stress, while another deletion mutant, pPcINO1.2.793 (of 793 bp), was significantly upregulated under salt stress. P. coarctata being a halophytic species, the high inducibility of pPcINO1.2.932 upon exposure to low-temperature stress was an unexpected result.


Assuntos
Mio-Inositol-1-Fosfato Sintase/genética , Proteínas de Plantas/genética , Poaceae/genética , Regiões Promotoras Genéticas/genética , Plantas Tolerantes a Sal/genética , Arabidopsis/genética , Oryza/enzimologia , Oryza/genética , Filogenia , Plantas Geneticamente Modificadas , Poaceae/enzimologia , Tolerância ao Sal/genética , Plantas Tolerantes a Sal/enzimologia , Tabaco/genética
12.
G3 (Bethesda) ; 8(9): 2913-2922, 2018 08 30.
Artigo em Inglês | MEDLINE | ID: mdl-29991509

RESUMO

Inositol is a precursor for the phospholipid membrane component phosphatidylinositol (PI), involved in signal transduction pathways, endoplasmic reticulum stress, and osmoregulation. Alterations of inositol metabolism have been implicated in human reproductive issues, the therapeutic effects of drugs used to treat epilepsy and bipolar disorder, spinal cord defects, and diseases including diabetes and Alzheimer's. The sole known inositol synthetic enzyme is myo-inositol synthase (MIPS), and the homolog in Drosophilia melanogaster is encoded by the Inos gene. Three identical deletion strains (inosΔDF /CyO) were constructed, confirmed by PCR and sequencing, and homozygotes (inosΔDF /inosΔDF ) were shown to lack the transcript encoding the MIPS enzyme. Without inositol, homozygous inosΔDF deletion fertilized eggs develop only to the first-instar larval stage. When transferred as pupae to food without inositol, however, inosΔDF homozygotes die significantly sooner than wild-type flies. Even with dietary inositol the homozygous inosΔDF males are sterile. An inos allele, with a P-element inserted into the first intron, fails to complement this male sterile phenotype. An additional copy of the Inos gene inserted into another chromosome rescues all the phenotypes. These genetic and phenotypic analyses establish D. melanogaster as an excellent model organism in which to examine the role of inositol synthesis in development and reproduction.


Assuntos
Deleção de Genes , Infertilidade Masculina/genética , Íntrons , Mio-Inositol-1-Fosfato Sintase/genética , Animais , Drosophila melanogaster , Feminino , Infertilidade Masculina/enzimologia , Larva/enzimologia , Larva/genética , Masculino , Mio-Inositol-1-Fosfato Sintase/metabolismo
13.
Plant Mol Biol ; 97(3): 253-263, 2018 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-29777485

RESUMO

KEY MESSAGE: Co-suppressed MIPS2 transgenic lines allow bypass of the embryo lethal phenotype of the previously published triple knock-out and demonstrate the effects of MIPS on later stages of development. Regulation of inositol production is of interest broadly for its effects on plant growth and development. The enzyme L-myo-inositol 1-phosphate synthase (MIPS, also known as IPS) isomerizes D-glucose-6-P to D-inositol 3-P, and this is the rate-limiting step in inositol production. In Arabidopsis thaliana, the MIPS enzyme is encoded by three different genes, (AtMIPS1, AtMIPS2 and AtMIPS3), each of which has been shown to produce proteins with biochemically similar properties but differential expression patterns. Here, we report phenotypic and biochemical effects of MIPS co-suppression. We show that some plants engineered to overexpress MIPS2 in fact show reduced expression of AtMIPS1, AtMIPS2 and AtMIPS3, and show altered vegetative phenotype, reduced size and root length, and delayed flowering. Additionally, these plants show reduced inositol, increased glucose levels, and alteration of other metabolites. Our results suggest that the three AtMIPS genes work together to impact the overall synthesis of myo-inositol and overall inositol homeostasis.


Assuntos
Proteínas de Arabidopsis/metabolismo , Inositol/biossíntese , Mio-Inositol-1-Fosfato Sintase/metabolismo , Interferência de RNA , Arabidopsis/crescimento & desenvolvimento , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Regulação da Expressão Gênica de Plantas , Genes de Plantas , Homeostase , Metabolômica , Mio-Inositol-1-Fosfato Sintase/genética , Plantas Geneticamente Modificadas
14.
Enzyme Microb Technol ; 112: 1-5, 2018 May.
Artigo em Inglês | MEDLINE | ID: mdl-29499774

RESUMO

Myo-inositol (inositol) is important in the cosmetics, pharmaceutical and functional food industries. Here, we report a novel pathway to produce inositol from glucose by a trienzymatic cascade system involving polyphosphate glucokinase (PPGK), inositol 1-phosphate synthase (IPS) and inositol monophosphatase (IMP). The system contained three highly active enzymes, AspPPGK from Arthrobacter sp. OY3WO11, TbIPS from Trypanosoma brucei TREU927, and EcIMP from Escherichia coli. A trienzymatic cascade reaction was implemented, and the conversion ratio from glucose to inositol reached 90%, which is promising for the enzymatic synthesis of inositol without ATP supplementation.


Assuntos
Glucose/metabolismo , Inositol/biossíntese , Mio-Inositol-1-Fosfato Sintase/metabolismo , Monoéster Fosfórico Hidrolases/metabolismo , Fosfotransferases/metabolismo , Arthrobacter/enzimologia , Vias Biossintéticas , Biotecnologia , Escherichia coli/enzimologia , Cinética , Proteínas Recombinantes/metabolismo , Trypanosoma brucei brucei/enzimologia
15.
Tree Physiol ; 38(10): 1566-1577, 2018 10 01.
Artigo em Inglês | MEDLINE | ID: mdl-29579299

RESUMO

Myo-inositol is a vital compound in plants. As the key rate-limiting enzyme in myo-inositol biosynthesis, l-myo-inositol-1-phosphate synthase (MIPS) is regarded as a determinant of the myo-inositol content in plants. The up-regulation of MIPS genes can increase the myo-inositol content, thereby enhancing the plant's resistance to a variety of stresses. However, there are few reports on the roles of myo-inositol and the identification of MIPS in woody trees. In this study, a MIPS gene, named as PeMIPS1, was characterized from Populus euphratica Oliv. The heterologous expression of PeMIPS1 compensated for inositol production in the yeast inositol auxotrophic mutant ino1 and the phenotypic lesions of the atmips1-2 mutant, an Arabidopsis MIPS1 knock-out mutant. A subcellular location analysis showed that the PeMIPS1-GFP fusion was localized in the nucleus and cytoplasm, but not in the chloroplasts, indicating that PeMIPS1 represented the cytosolic form of MIPS in P. euphratica. Interestingly, PeMIPS1 was not only inducible by drought and high salinity, but also by CuSO4 treatment. The transgenic poplar lines overexpressing PeMIPS1 had greater plant heights, shoot biomasses and survival rates than the wild type during the salt- or copper-stress treatment, and this was accompanied by an increase in the myo-inositol content. The overexpression of PeMIPS1 resulted in the increased activities of antioxidant enzymes and the accumulation of ascorbate, a key nonenzymatic antioxidant in plant, which partly accounted for the enhanced reactive oxygen species-scavenging capacity and the lowered hydrogen peroxide and malondialdehyde levels in the transgenic poplar. To the best of our knowledge, this study is the first to report the roles of MIPS genes in the tolerance to copper stress.


Assuntos
Cobre/metabolismo , Regulação da Expressão Gênica de Plantas , Mio-Inositol-1-Fosfato Sintase/genética , Populus/fisiologia , Espécies Reativas de Oxigênio/metabolismo , Tolerância ao Sal , Arabidopsis/genética , Mio-Inositol-1-Fosfato Sintase/metabolismo , Plantas Geneticamente Modificadas/genética , Plantas Geneticamente Modificadas/fisiologia , Populus/genética , Saccharomyces cerevisiae/genética , Poluentes do Solo/metabolismo , Estresse Fisiológico
16.
J Biol Chem ; 292(45): 18713-18728, 2017 11 10.
Artigo em Inglês | MEDLINE | ID: mdl-28924045

RESUMO

In the yeast Saccharomyces cerevisiae, the Opi1p repressor controls the expression of INO1 via the Opi1p/Ino2p-Ino4p regulatory circuit. Inositol depletion favors Opi1p interaction with both Scs2p and phosphatidic acid at the endoplasmic reticulum (ER) membrane. Inositol supplementation, however, favors the translocation of Opi1p from the ER into the nucleus, where it interacts with the Ino2p-Ino4p complex, attenuating transcription of INO1 A strain devoid of Scs2p (scs2Δ) and a mutant, OPI1FFAT, lacking the ability to interact with Scs2p were utilized to examine the specific role(s) of the Opi1p-Scs2p interaction in the regulation of INO1 expression and overall lipid metabolism. Loss of the Opi1p-Scs2p interaction reduced INO1 expression and conferred inositol auxotrophy. Moreover, inositol depletion in strains lacking this interaction resulted in Opi1p being localized to sites of lipid droplet formation, coincident with increased synthesis of triacylglycerol. Supplementation of choline to inositol-depleted growth medium led to decreased TAG synthesis in all three strains. However, in strains lacking the Opi1p-Scs2p interaction, Opi1p remained in the nucleus, preventing expression of INO1 These data support the conclusion that a specific pool of phosphatidic acid, associated with lipid droplet formation in the perinuclear ER, is responsible for the initial rapid exit of Opi1p from the nucleus to the ER and is required for INO1 expression in the presence of choline. Moreover, the mitochondria-specific phospholipid, cardiolipin, was significantly reduced in both strains compromised for Opi1p-Scs2p interaction, indicating that this interaction is required for the transfer of phosphatidic acid from the ER to the mitochondria for cardiolipin synthesis.


Assuntos
Retículo Endoplasmático/metabolismo , Proteínas de Membrana/metabolismo , Membranas Mitocondriais/metabolismo , Mio-Inositol-1-Fosfato Sintase/metabolismo , Ácidos Fosfatídicos/metabolismo , Proteínas Repressoras/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Transporte Biológico , Cardiolipinas/metabolismo , Núcleo Celular/metabolismo , Colina/metabolismo , Deleção de Genes , Regulação Bacteriana da Expressão Gênica , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Gotículas Lipídicas , Metabolismo dos Lipídeos , Proteínas de Membrana/genética , Mutação , Mio-Inositol-1-Fosfato Sintase/genética , Fosforilação , Processamento de Proteína Pós-Traducional , Proteínas Recombinantes de Fusão/metabolismo , Proteínas Repressoras/genética , Saccharomyces cerevisiae/citologia , Saccharomyces cerevisiae/crescimento & desenvolvimento , Proteínas de Saccharomyces cerevisiae/genética
17.
PLoS One ; 12(9): e0185351, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-28950028

RESUMO

A molecular evolutionary analysis of a well conserved protein helps to determine the essential amino acids in the core catalytic region. Based on the chemical properties of amino acid residues, phylogenetic analysis of a total of 172 homologous sequences of a highly conserved enzyme, L-myo-inositol 1-phosphate synthase or MIPS from evolutionarily diverse organisms was performed. This study revealed the presence of six phylogenetically conserved blocks, out of which four embrace the catalytic core of the functional protein. Further, specific amino acid modifications targeting the lysine residues, known to be important for MIPS catalysis, were performed at the catalytic site of a MIPS from monocotyledonous model plant, Oryza sativa (OsMIPS1). Following this study, OsMIPS mutants with deletion or replacement of lysine residues in the conserved blocks were made. Based on the enzyme kinetics performed on the deletion/replacement mutants, phylogenetic and structural comparison with the already established crystal structures from non-plant sources, an evolutionarily conserved peptide stretch was identified at the active pocket which contains the two most important lysine residues essential for catalytic activity.


Assuntos
Evolução Biológica , Lisina/metabolismo , Mio-Inositol-1-Fosfato Sintase/metabolismo , Oligopeptídeos/metabolismo , Oryza/enzimologia , Sequência de Aminoácidos , Biocatálise , Domínio Catalítico , Cristalografia por Raios X , Mutagênese Sítio-Dirigida , Mio-Inositol-1-Fosfato Sintase/química , Oligopeptídeos/química , Oryza/genética , Filogenia , Homologia de Sequência de Aminoácidos
18.
PLoS One ; 12(8): e0182534, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-28817575

RESUMO

Myo-inositol, the precursor of all inositol compounds, is essential for the viability of eukaryotes. Identifying the factors that regulate inositol homeostasis is of obvious importance to understanding cell function and the pathologies underlying neurological and metabolic resulting from perturbation of inositol metabolism. The current study identifies Mck1, a GSK3 homolog, as a novel positive regulator of inositol de novo synthesis in yeast. Mck1 was required for normal activity of myo-inositol phosphate synthase (MIPS), which catalyzes the rate-limiting step of inositol synthesis. mck1Δ cells exhibited a 50% decrease in MIPS activity and a decreased rate of incorporation of [13C6]glucose into [13C6]-inositol-3-phosphate and [13C6]-inositol compared to WT cells. mck1Δ cells also exhibited decreased growth in the presence of the inositol depleting drug valproate (VPA), which was rescued by supplementation of inositol. However, in contrast to wild type cells, which exhibited more than a 40% decrease in MIPS activity in the presence of VPA, the drug did not significantly decrease MIPS activity in mck1Δ cells. These findings indicate that VPA-induced MIPS inhibition is Mck1-dependent, and suggest a model that unifies two current hypotheses of the mechanism of action of VPA-inositol depletion and GSK3 inhibition.


Assuntos
Antimaníacos/farmacologia , Inibidores Enzimáticos/farmacologia , Quinase 3 da Glicogênio Sintase/metabolismo , Inositol/metabolismo , Mio-Inositol-1-Fosfato Sintase/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Ácido Valproico/farmacologia , Quinase 3 da Glicogênio Sintase/genética , Mio-Inositol-1-Fosfato Sintase/antagonistas & inibidores , Saccharomyces cerevisiae/efeitos dos fármacos , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética
19.
Yeast ; 34(9): 383-395, 2017 09.
Artigo em Inglês | MEDLINE | ID: mdl-28581036

RESUMO

During exposure of yeast cells to low levels of hydrogen peroxide (H2 O2 ), the expression of several genes is regulated for cells to adapt to the surrounding oxidative environment. Such adaptation involves modification of plasma membrane lipid composition, reorganization of ergosterol-rich microdomains and altered gene expression of proteins involved in lipid and vesicle traffic, to decrease permeability to exogenous H2 O2 . Opi1p is a transcriptional repressor that is inactive when present at the nuclear membrane/endoplasmic reticulum, but represseses transcription of inositol upstream activating sequence (UASINO )-containing genes, many of which are involved in the synthesis of phospholipids and fatty acids, when it is translocated to the nucleus. We investigated whether H2 O2 in concentrations inducing adaptation regulates Opi1p function. We found that, in the presence of H2 O2 , GFP-Opi1p fusion protein translocates to the nucleus and, concomitantly, the expression of UASINO -containing genes is affected. We also investigated whether cysteine residues of Opi1p were implicated in the H2 O2 -mediated translocation of this protein to the nucleus and identified cysteine residue 159 as essential for this process. Our work shows that Opi1p is redox-regulated and establishes a new mechanism of gene regulation involving Opi1p, which is important for adaptation to H2 O2 in yeast cells. Copyright © 2017 John Wiley & Sons, Ltd.


Assuntos
Núcleo Celular/metabolismo , Retículo Endoplasmático/metabolismo , Regulação Fúngica da Expressão Gênica/efeitos dos fármacos , Peróxido de Hidrogênio/farmacologia , Proteínas Repressoras/genética , Proteínas de Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Adaptação Biológica , Fatores de Transcrição Hélice-Alça-Hélice Básicos/efeitos dos fármacos , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Ácidos Graxos/biossíntese , Peróxido de Hidrogênio/química , Concentração de Íons de Hidrogênio , Inositol/análise , Inositol/química , Microdomínios da Membrana/metabolismo , Proteínas de Transporte de Monossacarídeos/efeitos dos fármacos , Proteínas de Transporte de Monossacarídeos/genética , Mio-Inositol-1-Fosfato Sintase/efeitos dos fármacos , Mio-Inositol-1-Fosfato Sintase/genética , Oxirredução , Estresse Oxidativo , Permeabilidade , Fosfolipídeos/biossíntese , Saccharomyces cerevisiae/efeitos dos fármacos , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/efeitos dos fármacos
20.
Appl Microbiol Biotechnol ; 101(10): 4259-4268, 2017 May.
Artigo em Inglês | MEDLINE | ID: mdl-28243709

RESUMO

Kasugamycin (KSM), an aminoglycoside antibiotic isolated from Streptomyces kasugaensis cultures, has been used against rice blast disease for more than 50 years. We cloned the KSM biosynthetic gene (KBG) cluster from S. kasugaensis MB273-C4 and constructed three KBG cassettes (i.e., cassettes I-III) to enable heterologous production of KSM in many actinomycetes by constitutive expression of KBGs. Cassette I comprised all putative transcriptional units in the cluster, but it was placed under the control of the P neo promoter from Tn5. It was not maintained stably in Streptomyces lividans and did not transform Rhodococcus erythropolis. Cassette II retained the original arrangement of KBGs, except that the promoter of kasT, the specific activator gene for KBG, was replaced with P rpsJ , the constitutive promoter of rpsJ from Streptomyces avermitilis. To enhance the intracellular concentration of myo-inositol, an expression cassette of ino1 encoding the inositol-1-phosphate synthase from S. avermitilis was inserted into cassette II to generate cassette III. These two cassettes showed stable maintenance in S. lividans and R. erythropolis to produce KSM. Particularly, the transformants of S. lividans induced KSM production up to the same levels as those produced by S. kasugaensis. Furthermore, cassette III induced more KSM accumulation than cassette II in R. erythropolis, suggesting an exogenous supply of myo-inositol by the ino1 expression in the host. Cassettes II and III appear to be useful for heterologous KSM production in actinomycetes. Rhodococcus exhibiting a spherical form in liquid cultivation is also a promising heterologous host for antibiotic fermentation.


Assuntos
Aminoglicosídeos/biossíntese , Antibacterianos/biossíntese , Família Multigênica , Rhodococcus/genética , Streptomyces lividans/genética , Streptomyces/genética , Sequência de Bases , Clonagem Molecular , Fermentação , Regulação Bacteriana da Expressão Gênica , Genes Bacterianos , Inositol/biossíntese , Inositol/metabolismo , Mio-Inositol-1-Fosfato Sintase/genética , Mio-Inositol-1-Fosfato Sintase/metabolismo , Rhodococcus/metabolismo , Streptomyces/metabolismo , Fatores de Transcrição/metabolismo
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