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1.
Plant Physiol ; 2024 May 31.
Artigo em Inglês | MEDLINE | ID: mdl-38820143

RESUMO

ELONGATED HYPOCOTYL 5 (HY5) is a major light-associated transcription factor involved in plant growth and development. In Arabidopsis (Arabidopsis thaliana), the role of HY5 is very well-defined in regulating primary root growth and lateral root formation; however, information regarding its role in root hair development is still lacking, and little is known about the genetic pathways regulating this process. In this study, we investigated the role of HY5 and its associated components in root hair development. Detailed analysis of root hair phenotype in wild-type (WT) and light signaling mutants in light and dark conditions revealed the importance of light-dependent HY5-mediated root hair initiation. Altered auxin levels in the root apex of the hy5 mutant and interaction of HY5 with promoters of root hair developmental genes were responsible for differential expression of root hair developmental genes and phenotype in the hy5 mutant. The partial complementation of root hair in the hy5 mutant after external supplementation of auxin and regaining of root hair in PIN-FORMED 2 (pin2) and PIN-FORMED 2 (pin3) mutants after grafting suggested that the auxin-mediated root hair development pathway requires HY5. Furthermore, miR397b overexpression (miR397bOX) and CRISPR/Cas9-based mutants (miR397bCR) indicated miR397b targets genes encoding Reduced Residual Arabinose (RRA1/RRA2), which in turn regulate root hair growth. The regulation of the miR397b- (RRA1/RRA2) module by HY5 demonstrated its indirect role by targeting root hair cell wall genes. Together, this study demonstrated that HY5 controls root hair development by integrating auxin signalling and other miRNA-mediated pathways.

2.
Plant J ; 116(6): 1748-1765, 2023 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-37715733

RESUMO

The plant citrate transporters, functional in mineral nutrient uptake and homeostasis, usually belong to the multidrug and toxic compound extrusion transporter family. We identified and functionally characterized a rice (Oryza sativa) citrate transporter, OsCT1, which differs from known plant citrate transporters and is structurally close to rice silicon transporters. Domain analysis depicted that OsCT1 carries a bacterial citrate-metal transporter domain, CitMHS. OsCT1 showed citrate efflux activity when expressed in Xenopus laevis oocytes and is localized to the cell plasma membrane. It is highly expressed in the shoot and reproductive tissues of rice, and its promoter activity was visible in cells surrounding the vasculature. The OsCT1 knockout (KO) lines showed a reduced citrate content in the shoots and the root exudates, whereas overexpression (OE) line showed higher citrate exudation from their roots. Further, the KO and OE lines showed variations in the manganese (Mn) distribution leading to changes in their agronomical traits. Under deficient conditions (Mn-sufficient conditions followed by 8 days of 0 µm MnCl2 · 4H2 O treatment), the supply of manganese towards the newer leaf was found to be obstructed in the KO line. There were no significant differences in phosphorus (P) distribution; however, P uptake was reduced in the KO and increased in OE lines at the vegetative stage. Further, experiments in Xenopus oocytes revealed that OsCT1 could efflux citrate with Mn. In this way, we provide insights into a mechanism of citrate-metal transport in plants and its role in mineral homeostasis, which remains conserved with their bacterial counterparts.


Assuntos
Oryza , Oryza/genética , Oryza/metabolismo , Manganês/metabolismo , Fósforo/metabolismo , Proteínas de Membrana Transportadoras/metabolismo , Ácido Cítrico/metabolismo , Minerais/metabolismo , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Raízes de Plantas/genética , Raízes de Plantas/metabolismo , Regulação da Expressão Gênica de Plantas
3.
J Exp Bot ; 73(14): 5033-5051, 2022 08 11.
Artigo em Inglês | MEDLINE | ID: mdl-35526193

RESUMO

Galactolipids are essential to compensate for the loss of phospholipids by 'membrane lipid remodelling' in plants under phosphorus (P) deficiency conditions. Monogalactosyl diacylglycerol (MGDG) synthases catalyse the synthesis of MGDG which is further converted into digalactosyl diacylglycerol (DGDG), later replacing phospholipids in the extraplastidial membranes. However, the roles of these enzymes are not well explored in rice. In this study, the rice MGDG synthase 3 gene (OsMGD3) was identified and functionally characterized. We showed that the plant phosphate (Pi) status and the transcription factor PHOSPHATE STARVATION RESPONSE 2 (OsPHR2) are involved in the transcriptional regulation of OsMGD3. CRISPR/Cas9 knockout and overexpression lines of OsMGD3 were generated to explore its potential role in rice adaptation to Pi deficiency. Compared with the wild type, OsMGD3 knockout lines displayed a reduced Pi acquisition and utilization while overexpression lines showed an enhancement of the same. Further, OsMGD3 showed a predominant role in roots, altering lateral root growth. Our comprehensive lipidomic analysis revealed a role of OsMGD3 in membrane lipid remodelling, in addition to a role in regulating diacylglycerol and phosphatidic acid contents that affected the expression of Pi transporters. Our study highlights the role of OsMGD3 in affecting both internal P utilization and P acquisition in rice.


Assuntos
Oryza , Diglicerídeos/metabolismo , Galactosiltransferases , Lipídeos de Membrana/metabolismo , Oryza/metabolismo , Fosfatos/metabolismo , Fosfolipídeos/metabolismo , Plantas/metabolismo
4.
Biochem Biophys Res Commun ; 605: 51-55, 2022 05 21.
Artigo em Inglês | MEDLINE | ID: mdl-35316763

RESUMO

microRNAs (miRNAs) regulate target gene expression by pairing to target mRNAs, leading to mRNA degradation or translation inhibition. Out of several miRNAs in Arabidopsis, miR397b and miR857 regulate secondary growth by modulating lignin polymerization and deposition in secondary xylem cells by targeting laccases. Interestingly, the phytohormone ethylene is also suggested to have a role in lignin biosynthesis in tension wood formation. Despite this information, it is not known whether ethylene has any role in controlling secondary growth via miRNAs-mediated pathways. In this study, we elucidate that ethylene acts upstream to the miR397b/miR857-laccases module and negatively regulates lignin biosynthesis by directly activating the expression of both the miRNAs. The binding of EIN3 to the promoter of miR397b is further validated by yeast one-hybrid assay. In addition to its role in lignification, ethylene also regulates leaf serration by directly regulating the expression of NAC transcription factors, like CUP-SHAPED COTYLEDON2 (CUC2) and CUC3. Together, our study suggests a novel mechanism involving ethylene and miRNAs in lignin biosynthesis and leaf serration in Arabidopsis thaliana.


Assuntos
Proteínas de Arabidopsis , Arabidopsis , MicroRNAs , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Etilenos , Regulação da Expressão Gênica de Plantas , Lignina/metabolismo , MicroRNAs/genética , MicroRNAs/metabolismo , Folhas de Planta/metabolismo
5.
Plant Sci ; 313: 111073, 2021 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-34763865

RESUMO

MicroRNAs (miRNAs), a class of single-stranded non-coding RNA of 20-24 nucleotides, regulate gene expression by target gene transcript cleavage or translation inhibition. The phytohormone auxin is a crucial regulator of almost every process involved in plant growth and development. Several studies have demonstrated the involvement of miRNA(s) in the regulation of the auxin signaling pathway and plant development. However, very few studies have identified the auxin-mediated regulation of miRNA(s). In this study, we reveal the detailed mechanism of auxin-mediated regulation of the cell wall-related miR775- Galactosyl transferase (GalT) module, which plays an important role in root growth in Arabidopsis thaliana. We also showed two interdependent mechanisms by which miR775 regulates root growth: miR775-GalT and light-mediated sucrose-dependent pathways. Treatment of GUS reporter lines with Indole Acetic Acid (IAA), sucrose, and light apparently enhanced the abundance of miR775 in root tissue. miR775 overexpressing (miR775OX) lines showed changes in root architecture, including increased primary root growth and root hair, by targeting GalT. miR775OX lines also showed tolerance toward low Pi. These results provide new insights into the auxin regulation of cell wall-related miR775 and suggest its significant role in plant root growth and development by modifying the cell wall.


Assuntos
Arabidopsis/crescimento & desenvolvimento , Arabidopsis/genética , Ácidos Indolacéticos/metabolismo , MicroRNAs/metabolismo , Raízes de Plantas/crescimento & desenvolvimento , Raízes de Plantas/genética , Sacarose/metabolismo , Adaptação Ocular/efeitos dos fármacos , Adaptação Ocular/genética , Regulação da Expressão Gênica de Plantas , Variação Genética , Genótipo , Crescimento e Desenvolvimento/genética , Redes e Vias Metabólicas/genética
6.
Plant Sci ; 303: 110760, 2021 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-33487344

RESUMO

Flavonols, a class of flavonoids, accumulate as protective agents in response to various stresses. Among various environmental stimuli, light is one of the factors regulating flavonol production. MYB12/11/111, members of the R2R3 MYBs family, regulates spatio-temporal flavonol accumulation in Arabidopsis. Although various studies indicate at the involvement of an E3 ubiquitin ligase CONSTITUTIVE PHOTOMORPHOGENIC 1 (COP1) and ELONGATED HYPOCOTYL 5 (HY5) in flavonoid biosynthesis in response to UV-B, the regulatory roles of these components under visible light are yet to be investigated. Here, we demonstrate that flavonol accumulation in Arabidopsis is light-regulated. Furthermore, our analysis suggests that MYB12 is a HY5-dependent light-inducible gene and plays a key role in the activation of the flavonol biosynthesis in response to light. Our results indicate the involvement of COP1 in the dark-dependent repression of MYB12 expression and flavonol accumulation. In addition, results also suggest that the effect of COP1 on MYB12 is indirect and is mediated through HY5, a direct transcriptional activator of the MYB12. Together these findings indicate that COP1 acts as a master negative regulator of flavonol biosynthesis in the dark.


Assuntos
Proteínas de Arabidopsis/fisiologia , Arabidopsis/metabolismo , Fatores de Transcrição de Zíper de Leucina Básica/fisiologia , Flavonóis/biossíntese , Ubiquitina-Proteína Ligases/fisiologia , Proteínas de Arabidopsis/metabolismo , Fatores de Transcrição de Zíper de Leucina Básica/metabolismo , Regulação da Expressão Gênica de Plantas/efeitos da radiação , Luz , Plantas Geneticamente Modificadas , Reação em Cadeia da Polimerase em Tempo Real , Fatores de Transcrição/metabolismo , Fatores de Transcrição/fisiologia , Técnicas do Sistema de Duplo-Híbrido , Ubiquitina-Proteína Ligases/metabolismo , Raios Ultravioleta
7.
Nat Plants ; 6(10): 1262-1274, 2020 10.
Artigo em Inglês | MEDLINE | ID: mdl-32958895

RESUMO

MicroRNAs (miRNAs) are processed products of primary miRNAs (pri-miRNAs) and regulate the target gene expression. Though the regulatory roles of the several mature plant miRNAs have been studied in detail, the functions of other regions of the pri-miRNAs are still unrecognized. Recent studies suggest that a few pri-miRNAs may encode small peptides, miRNA-encoded peptides (miPEPs); however, the functions of these peptides have not been studied in detail. We report that the pri-miR858a of Arabidopsis thaliana encodes a small peptide, miPEP858a, which regulates the expression of pri-miR858a and associated target genes. miPEP858a-edited and miPEP858a-overexpressing lines showed altered plant development and accumulated modulated levels of flavonoids due to changes in the expression of genes associated with the phenylpropanoid pathway and auxin signalling. The exogenous treatment of the miPEP858a-edited plants with synthetic miPEP858a complemented the phenotypes and the gene function. This study suggests the importance of miPEP858a in exerting control over plant development and the phenylpropanoid pathway.


Assuntos
Proteínas de Arabidopsis/genética , Arabidopsis/genética , Flavonoides/biossíntese , MicroRNAs/genética , Peptídeos/genética , RNA de Plantas/genética , Antocianinas/metabolismo , Arabidopsis/crescimento & desenvolvimento , Regulação da Expressão Gênica de Plantas , Ácidos Indolacéticos/metabolismo , Lignina/metabolismo , Mutação , Fenótipo , Desenvolvimento Vegetal/genética , RNA de Plantas/metabolismo
8.
Biochem J ; 477(8): 1525-1539, 2020 04 30.
Artigo em Inglês | MEDLINE | ID: mdl-32242624

RESUMO

Nek7 is a serine/threonine-protein kinase required for proper spindle formation and cytokinesis. Elevated Nek7 levels have been observed in several cancers, and inhibition of Nek7 might provide a route to the development of cancer therapeutics. To date, no selective and potent Nek7 inhibitors have been identified. Nek7 crystal structures exhibit an improperly formed regulatory-spine (R-spine), characteristic of an inactive kinase. We reasoned that the preference of Nek7 to crystallise in this inactive conformation might hinder attempts to capture Nek7 in complex with Type I inhibitors. Here, we have introduced aromatic residues into the R-spine of Nek7 with the aim to stabilise the active conformation of the kinase through R-spine stacking. The strong R-spine mutant Nek7SRS retained catalytic activity and was crystallised in complex with compound 51, an ATP-competitive inhibitor of Nek2 and Nek7. Subsequently, we obtained the same crystal form for wild-type Nek7WT in apo form and bound to compound 51. The R-spines of the three well-ordered Nek7WT molecules exhibit variable conformations while the R-spines of the Nek7SRS molecules all have the same, partially stacked configuration. Compound 51 bound to Nek2 and Nek7 in similar modes, but differences in the precise orientation of a substituent highlights features that could be exploited in designing inhibitors that are selective for particular Nek family members. Although the SRS mutations are not required to obtain a Nek7-inhibitor structure, we conclude that it is a useful strategy for restraining the conformation of a kinase in order to promote crystallogenesis.


Assuntos
Inibidores Enzimáticos/metabolismo , Quinases Relacionadas a NIMA/química , Quinases Relacionadas a NIMA/metabolismo , Catálise , Inibidores Enzimáticos/química , Humanos , Cinética , Mutação , Quinases Relacionadas a NIMA/genética , Ligação Proteica , Conformação Proteica , Engenharia de Proteínas
9.
J Med Chem ; 62(5): 2447-2465, 2019 03 14.
Artigo em Inglês | MEDLINE | ID: mdl-30779566

RESUMO

A series of imidazo[1,2- b]pyridazin-8-amine kinase inhibitors were discovered to allosterically inhibit the endoribonuclease function of the dual kinase-endoribonuclease inositol-requiring enzyme 1α (IRE1α), a key component of the unfolded protein response in mammalian cells and a potential drug target in multiple human diseases. Inhibitor optimization gave compounds with high kinome selectivity that prevented endoplasmic reticulum stress-induced IRE1α oligomerization and phosphorylation, and inhibited endoribonuclease activity in human cells. X-ray crystallography showed the inhibitors to bind to a previously unreported and unusually disordered conformation of the IRE1α kinase domain that would be incompatible with back-to-back dimerization of the IRE1α protein and activation of the endoribonuclease function. These findings increase the repertoire of known IRE1α protein conformations and can guide the discovery of highly selective ligands for the IRE1α kinase site that allosterically inhibit the endoribonuclease.


Assuntos
Endorribonucleases/antagonistas & inibidores , Endorribonucleases/metabolismo , Inibidores de Proteínas Quinases/farmacologia , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Proteínas Serina-Treonina Quinases/metabolismo , Regulação Alostérica , Biopolímeros/metabolismo , Cristalografia por Raios X , Dimerização , Estresse do Retículo Endoplasmático/efeitos dos fármacos , Endorribonucleases/química , Células HEK293 , Humanos , Fosforilação , Conformação Proteica , Inibidores de Proteínas Quinases/metabolismo , Proteínas Serina-Treonina Quinases/química
10.
Plant Cell Physiol ; 59(10): 2099-2112, 2018 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-30010959

RESUMO

Plants are continuously exposed to a myriad of stresses, which lead to the formation of secondary metabolites including flavonoids. Studies suggest that low temperature exposure leads to enhanced flavonoid accumulation in Arabidopsis thaliana. In addition, flavonoid biosynthesis is regulated by light through various regulatory factors. Therefore, plants may possess the capability to integrate light and low temperature signals for survival under freezing conditions. However, the detailed molecular mechanism and the regulatory factors associated with light- and low temperature- responsive flavonoid biosynthesis remain largely unknown. Here, we report a strict requirement for light for the low temperature-enhanced flavonol biosynthesis. Low temperature-induced expression of biosynthetic genes as well as flavonol accumulation was hampered in ELONGATED HYPOCOTYL (hy5) and myb11myb111myb12 triple mutants as compared with the wild type in Arabidopsis. Overexpression of AtHY5 in the hy5 mutant restored induction of gene expression and flavonol accumulation in response to low temperature in light. Metabolite and gene expression analysis also suggests a negative role for CONSTITUTIVE PHOTOMORPHOGENIC1 (COP1) in accumulation of flavonols in response to low temperature. Overexpression of AtMYB12 enhanced accumulation of flavonols under low temperature in a light-dependent manner. Together, our analysis suggests the requirement for HY5 and flavonol-specific MYB regulatory factors for low temperature-induced flavonol synthesis.


Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Flavonóis/metabolismo , Arabidopsis/fisiologia , Proteínas de Arabidopsis/fisiologia , Regulação da Expressão Gênica de Plantas/genética , Regulação da Expressão Gênica de Plantas/fisiologia , Hipocótilo/metabolismo , Hipocótilo/fisiologia , Temperatura , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo
11.
Plant Physiol ; 171(2): 944-59, 2016 06.
Artigo em Inglês | MEDLINE | ID: mdl-27208307

RESUMO

MicroRNAs (miRNAs) are endogenous, noncoding small RNAs that function as critical regulators of gene expression. In plants, miRNAs have shown their potential as regulators of growth, development, signal transduction, and stress tolerance. Although the miRNA-mediated regulation of several processes is known, the involvement of miRNAs in regulating secondary plant product biosynthesis is poorly understood. In this study, we functionally characterized Arabidopsis (Arabidopsis thaliana) miR858a, which putatively targets R2R3-MYB transcription factors involved in flavonoid biosynthesis. Overexpression of miR858a in Arabidopsis led to the down-regulation of several MYB transcription factors regulating flavonoid biosynthesis. In contrast to the robust growth and early flowering of miR858OX plants, reduction of plant growth and delayed flowering were observed in Arabidopsis transgenic lines expressing an artificial miRNA target mimic (MIM858). Genome-wide expression analysis using transgenic lines suggested that miR858a targets a number of regulatory factors that modulate the expression of downstream genes involved in plant development and hormonal and stress responses. Furthermore, higher expression of MYBs in MIM858 lines leads to redirection of the metabolic flux towards the synthesis of flavonoids at the cost of lignin synthesis. Altogether, our study has established the potential role of light-regulated miR858a in flavonoid biosynthesis and plant growth and development.


Assuntos
Proteínas de Arabidopsis/genética , Arabidopsis/genética , Flavonoides/metabolismo , Regulação da Expressão Gênica de Plantas , MicroRNAs/metabolismo , Fatores de Transcrição/genética , Arabidopsis/crescimento & desenvolvimento , Arabidopsis/metabolismo , Proteínas de Arabidopsis/metabolismo , Vias Biossintéticas , Regulação para Baixo , Flores/genética , Flores/crescimento & desenvolvimento , Flores/metabolismo , Metabolômica , MicroRNAs/genética , Fenótipo , Folhas de Planta/genética , Folhas de Planta/crescimento & desenvolvimento , Folhas de Planta/metabolismo , Plantas Geneticamente Modificadas , Plântula/genética , Plântula/crescimento & desenvolvimento , Plântula/metabolismo , Sementes/genética , Sementes/crescimento & desenvolvimento , Sementes/metabolismo , Fatores de Transcrição/metabolismo
12.
Sci Rep ; 6: 21668, 2016 Feb 26.
Artigo em Inglês | MEDLINE | ID: mdl-26917158

RESUMO

Externally visible body and longitudinal bone growth is a result of proliferation of chondrocytes. In growth disorder, there is delay in the age associated increase in height. The present study evaluates the effect of extract from transgenic tomato fruit expressing AtMYB12 transcription factor on bone health including longitudinal growth. Constitutive expression of AtMYB12 in tomato led to a significantly enhanced biosynthesis of flavonoids in general and the flavonol biosynthesis in particular. Pre-pubertal ovary intact BALB/c mice received daily oral administration of vehicle and ethanolic extract of wild type (WT-TOM) and transgenic AtMYB12-tomato (MYB12-TOM) fruits for six weeks. Animal fed with MYB12-TOM showed no inflammation in hepatic tissues and normal sinusoidal Kupffer cell morphology. MYB12-TOM extract significantly increased tibial and femoral growth and subsequently improved the bone length as compared to vehicle and WT-TOM. Histomorphometry exhibited significantly wider distal femoral and proximal tibial growth plate, increased number and size of hypertrophic chondrocytes in MYB12-TOM which corroborated with micro-CT and expression of BMP-2 and COL-10, marker genes for hypertrophic cells. We conclude that metabolic reprogramming of tomato by AtMYB12 has the potential to improve longitudinal bone growth thus helping in achievement of greater peak bone mass during adolescence.


Assuntos
Proteínas de Arabidopsis , Condrogênese/efeitos dos fármacos , Flavonóis/farmacologia , Solanum lycopersicum/química , Fatores de Transcrição , Animais , Feminino , Flavonóis/biossíntese , Frutas/química , Solanum lycopersicum/genética , Solanum lycopersicum/metabolismo , Camundongos , Plantas Geneticamente Modificadas/química , Plantas Geneticamente Modificadas/metabolismo
13.
Oncotarget ; 6(15): 13019-35, 2015 May 30.
Artigo em Inglês | MEDLINE | ID: mdl-25968568

RESUMO

IRE1 transduces the unfolded protein response by splicing XBP1 through its C-terminal cytoplasmic kinase-RNase region. IRE1 autophosphorylation is coupled to RNase activity through formation of a back-to-back dimer, although the conservation of the underlying molecular mechanism is not clear from existing structures. We have crystallized human IRE1 in a back-to-back conformation only previously seen for the yeast homologue. In our structure the kinase domain appears primed for catalysis but the RNase domains are disengaged. Structure-function analysis reveals that IRE1 is autoinhibited through a Tyr-down mechanism related to that found in the unrelated Ser/Thr protein kinase Nek7. We have developed a compound that potently inhibits human IRE1 kinase activity while stimulating XBP1 splicing. A crystal structure of the inhibitor bound to IRE1 shows an increased ordering of the kinase activation loop. The structures of hIRE in apo and ligand-bound forms are consistent with a previously proposed model of IRE1 regulation in which formation of a back-to-back dimer coupled to adoption of a kinase-active conformation drive RNase activation. The structures provide opportunities for structure-guided design of IRE1 inhibitors.


Assuntos
Endorribonucleases/química , Endorribonucleases/metabolismo , Proteínas Serina-Treonina Quinases/química , Proteínas Serina-Treonina Quinases/metabolismo , Descoberta de Drogas , Endorribonucleases/antagonistas & inibidores , Endorribonucleases/genética , Humanos , Ligantes , Modelos Moleculares , Fosforilação , Conformação Proteica , Inibidores de Proteínas Quinases/química , Inibidores de Proteínas Quinases/farmacologia , Multimerização Proteica , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Proteínas Serina-Treonina Quinases/genética , Ribonucleases/genética , Ribonucleases/metabolismo , Relação Estrutura-Atividade , Transfecção
14.
Chem Biol Interact ; 234: 114-25, 2015 Jun 05.
Artigo em Inglês | MEDLINE | ID: mdl-25526675

RESUMO

Short-chain dehydrogenases/reductases (SDRs) constitute a large, functionally diverse branch of enzymes within the class of NAD(P)(H) dependent oxidoreductases. In humans, over 80 genes have been identified with distinct metabolic roles in carbohydrate, amino acid, lipid, retinoid and steroid hormone metabolism, frequently associated with inherited genetic defects. Besides metabolic functions, a subset of atypical SDR proteins appears to play critical roles in adapting to redox status or RNA processing, and thereby controlling metabolic pathways. Here we present an update on the human SDR superfamily and a ligand identification strategy using differential scanning fluorimetry (DSF) with a focused library of oxidoreductase and metabolic ligands to identify substrate classes and inhibitor chemotypes. This method is applicable to investigate structure-activity relationships of oxidoreductases and ultimately to better understand their physiological roles.


Assuntos
Ácido Graxo Sintases/genética , Ácido Graxo Sintases/metabolismo , NADH NADPH Oxirredutases/genética , NADH NADPH Oxirredutases/metabolismo , Humanos , Ligantes , Oxirredutases/genética , Oxirredutases/metabolismo , Filogenia , Relação Estrutura-Atividade , Especificidade por Substrato/genética
15.
Sci Rep ; 4: 5018, 2014 May 21.
Artigo em Inglês | MEDLINE | ID: mdl-24846090

RESUMO

Flavonoids, due to their pharmacological attributes, have recently become target molecules for metabolic engineering in commonly consumed food crops. Strategies including expression of single genes and gene pyramiding have provided only limited success, due principally to the highly branched and complex biosynthetic pathway of the flavonoids. Transcription factors have been demonstrated as an efficient tool for metabolic engineering of this pathway, but often exhibit variation in heterologous systems relative to that in the homologous system. In the present work, Arabidopsis MYB transcription factor, AtMYB111, has been expressed in tobacco to study whether this can enhance flavonoid biosynthesis in heterologous system. The results suggest that AtMYB111 expression in transgenic tobacco enhances expression of genes of the phenylpropanoid pathway leading to an elevated content of flavonols. However, dark incubation of transgenic and wild type (WT) plants down-regulated both the expression of genes as well as flavonoid content as compared to light grown plants. The study concludes that AtMYB111 can be effectively used in heterologous systems, however, light is required for its action in modulating biosynthetic pathway.


Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Vias Biossintéticas/efeitos da radiação , Flavonóis/metabolismo , Regulação da Expressão Gênica de Plantas/efeitos da radiação , Luz , Nicotiana/metabolismo , Plantas Geneticamente Modificadas/metabolismo , Fatores de Transcrição/metabolismo , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Cromatografia Líquida , Plantas Geneticamente Modificadas/genética , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Nicotiana/genética , Fatores de Transcrição/genética
16.
Appl Radiat Isot ; 70(7): 1254-60, 2012 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-22204785

RESUMO

For the design and modeling of Accelerator Driven sub-critical System (ADS) a detailed study of response of ADS materials to the spallation neutrons is required. For this purpose reaction rates of different reactions like (n, xn) and (n, xnyp) in 209Bi, natMo, 56Fe, natNi, 55Mn, natTi and natCo materials are determined in an experiment conducted at Nuclotron of JINR, Dubna, using 1.6 GeV d-beam in the 'Energy+Transmutation' set-up. Reaction rates of various (n, xn) and (n, xnyp) reactions are studied in these samples. Results of reaction rates deduced from all the gamma peaks observed in case of 209Bi (n, xn) reactions with x=3-9, natMo (n, γ), (n, 3n), (n, 6n), 56Fe (n, p), (n, p2n), (n, p4n), natNi (n, 2n), (n, 3n), (n, p), (n, d), (n, t), 55Mn (n, γ), (n, 2n), (n, 4n), natTi (n, p), (n, d), (n, t) and natCo (n, γ), (n, xn) reactions with x=2-5 along with (n, p), (n, 2p2n), (n, 2p4n) and (n, 2p6n) are presented. The measured reaction rates for all the elements show good consistency for all the reaction channels and all observed Eγ's of the product nucleus. For all the above mentioned reactions, both experimental as well as theoretical spectrum average cross-sections (σsp.av.cs) are deduced and compared. A close agreement is found between the experimental σsp.av.cs and theoretical σsp.av.cs values.

17.
PLoS One ; 5(11): e13719, 2010 Nov 02.
Artigo em Inglês | MEDLINE | ID: mdl-21072165

RESUMO

BACKGROUND: 15-Hydroxyprostaglandin dehydrogenase (15-PGDH, EC 1.1.1.141) is the key enzyme for the inactivation of prostaglandins, regulating processes such as inflammation or proliferation. The anabolic pathways of prostaglandins, especially with respect to regulation of the cyclooxygenase (COX) enzymes have been studied in detail; however, little is known about downstream events including functional interaction of prostaglandin-processing and -metabolizing enzymes. High-affinity probes for 15-PGDH will, therefore, represent important tools for further studies. PRINCIPAL FINDINGS: To identify novel high-affinity inhibitors of 15-PGDH we performed a quantitative high-throughput screen (qHTS) by testing >160 thousand compounds in a concentration-response format and identified compounds that act as noncompetitive inhibitors as well as a competitive inhibitor, with nanomolar affinity. Both types of inhibitors caused strong thermal stabilization of the enzyme, with cofactor dependencies correlating with their mechanism of action. We solved the structure of human 15-PGDH and explored the binding modes of the inhibitors to the enzyme in silico. We found binding modes that are consistent with the observed mechanisms of action. CONCLUSIONS: Low cross-reactivity in screens of over 320 targets, including three other human dehydrogenases/reductases, suggest selectivity of the present inhibitors for 15-PGDH. The high potencies and different mechanisms of action of these chemotypes make them a useful set of complementary chemical probes for functional studies of prostaglandin-signaling pathways. ENHANCED VERSION: This article can also be viewed as an enhanced version in which the text of the article is integrated with interactive 3D representations and animated transitions. Please note that a web plugin is required to access this enhanced functionality. Instructions for the installation and use of the web plugin are available in Text S2.


Assuntos
Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacologia , Hidroxiprostaglandina Desidrogenases/antagonistas & inibidores , Hidroxiprostaglandina Desidrogenases/química , Sítios de Ligação , Ligação Competitiva , Biocatálise/efeitos dos fármacos , Cristalografia por Raios X , Dinoprostona/metabolismo , Relação Dose-Resposta a Droga , Inibidores Enzimáticos/metabolismo , Humanos , Hidroxiprostaglandina Desidrogenases/metabolismo , Cinética , Modelos Moleculares , Estrutura Molecular , NAD/química , NAD/metabolismo , Oxirredução , Ligação Proteica , Multimerização Proteica , Estrutura Terciária de Proteína , Relação Estrutura-Atividade , Especificidade por Substrato
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