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1.
Nat Chem Biol ; 8(5): 477-85, 2012 Apr 01.
Artigo em Inglês | MEDLINE | ID: mdl-22466420

RESUMO

The plant hormone auxin regulates virtually every aspect of plant growth and development. Auxin acts by binding the F-box protein transport inhibitor response 1 (TIR1) and promotes the degradation of the AUXIN/INDOLE-3-ACETIC ACID (Aux/IAA) transcriptional repressors. Here we show that efficient auxin binding requires assembly of an auxin co-receptor complex consisting of TIR1 and an Aux/IAA protein. Heterologous experiments in yeast and quantitative IAA binding assays using purified proteins showed that different combinations of TIR1 and Aux/IAA proteins form co-receptor complexes with a wide range of auxin-binding affinities. Auxin affinity seems to be largely determined by the Aux/IAA. As there are 6 TIR1/AUXIN SIGNALING F-BOX proteins (AFBs) and 29 Aux/IAA proteins in Arabidopsis thaliana, combinatorial interactions may result in many co-receptors with distinct auxin-sensing properties. We also demonstrate that the AFB5-Aux/IAA co-receptor selectively binds the auxinic herbicide picloram. This co-receptor system broadens the effective concentration range of the hormone and may contribute to the complexity of auxin response.


Assuntos
Proteínas de Arabidopsis/química , Proteínas de Ligação a DNA/química , Proteínas F-Box/química , Ácidos Indolacéticos/química , Proteínas Nucleares/química , Receptores de Superfície Celular/química , Sequência de Aminoácidos , Herbicidas/química , Dados de Sequência Molecular , Picloram/química
2.
Plant J ; 65(6): 980-90, 2011 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-21323771

RESUMO

The use of small molecules has great power to dissect biological processes. This study presents the identification and characterisation of an inhibitor of peroxisome matrix protein import. A mini-screen was carried out to identify molecules that cause alteration in peroxisome morphology, or mislocalization of a peroxisome targeted fluorescent reporter protein. A benzimidazole lead compound (LDS-003655) was identified that resulted in reduced GFP fluorescence in peroxisomes and cytosolic GFP accumulation. The effect of the compound was specific to peroxisomes as Golgi bodies, endoplasmic reticulum and the actin cytoskeleton were unaffected even at 25 µM, whereas peroxisome import via the PTS1 pathway was compromised at 100 nM. When seedlings were grown on 25 µM LDS-003655 they displayed morphology typical of seedlings grown in the presence of auxin, and expression of the auxin reporter DR5::GFP was induced. Analysis of a focussed library of LDS-003655 derivatives in comparison with known auxins led to the conclusion that the auxin-like activity of LDS-003655 is attributable to its in situ hydrolysis giving rise to 2,5-dichlorobenzoic acid, whereas the import inhibiting activity of LDS-003655 requires the whole molecule. None of the auxins tested had any effect on peroxisome protein import. Matrix import by the PTS2 import pathway was relatively insensitive to LDS-003655 and its active analogues, with effects only seen after prolonged incubation on high concentrations. Steady-state protein levels of PEX5, the PTS1 import pathway receptor, were reduced in the presence of 100 nM LDS-003655, suggesting a possible mechanism for the import inhibition.


Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/efeitos dos fármacos , Arabidopsis/metabolismo , Benzimidazóis/farmacologia , Peroxissomos/efeitos dos fármacos , Peroxissomos/metabolismo , Receptores Citoplasmáticos e Nucleares/metabolismo , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Sequência de Bases , Benzimidazóis/química , Transporte Biológico Ativo/efeitos dos fármacos , DNA de Plantas/genética , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Ácidos Indolacéticos/farmacologia , Receptor 2 de Sinal de Orientação para Peroxissomos , Receptor 1 de Sinal de Orientação para Peroxissomos , Reguladores de Crescimento de Plantas/farmacologia , Plantas Geneticamente Modificadas , Receptores Citoplasmáticos e Nucleares/genética , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Transdução de Sinais/efeitos dos fármacos , Relação Estrutura-Atividade
3.
Mol Syst Biol ; 7: 508, 2011 Jul 05.
Artigo em Inglês | MEDLINE | ID: mdl-21734647

RESUMO

The plant hormone auxin is thought to provide positional information for patterning during development. It is still unclear, however, precisely how auxin is distributed across tissues and how the hormone is sensed in space and time. The control of gene expression in response to auxin involves a complex network of over 50 potentially interacting transcriptional activators and repressors, the auxin response factors (ARFs) and Aux/IAAs. Here, we perform a large-scale analysis of the Aux/IAA-ARF pathway in the shoot apex of Arabidopsis, where dynamic auxin-based patterning controls organogenesis. A comprehensive expression map and full interactome uncovered an unexpectedly simple distribution and structure of this pathway in the shoot apex. A mathematical model of the Aux/IAA-ARF network predicted a strong buffering capacity along with spatial differences in auxin sensitivity. We then tested and confirmed these predictions using a novel auxin signalling sensor that reports input into the signalling pathway, in conjunction with the published DR5 transcriptional output reporter. Our results provide evidence that the auxin signalling network is essential to create robust patterns at the shoot apex.


Assuntos
Arabidopsis/crescimento & desenvolvimento , Ácidos Indolacéticos/farmacologia , Reguladores de Crescimento de Plantas/farmacologia , Brotos de Planta/crescimento & desenvolvimento , Transdução de Sinais/genética , Arabidopsis/genética , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Análise por Conglomerados , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Regulação da Expressão Gênica de Plantas , Genes de Plantas , Hibridização in Situ Fluorescente , Meristema/química , Meristema/metabolismo , Microscopia Confocal , Modelos Teóricos , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Organogênese , Brotos de Planta/genética , Brotos de Planta/metabolismo , Plantas Geneticamente Modificadas , Transcrição Gênica
4.
ACS Chem Biol ; 9(3): 673-82, 2014 Mar 21.
Artigo em Inglês | MEDLINE | ID: mdl-24313839

RESUMO

Structure-activity profiles for the phytohormone auxin have been collected for over 70 years, and a number of synthetic auxins are used in agriculture. Auxin classification schemes and binding models followed from understanding auxin structures. However, all of the data came from whole plant bioassays, meaning the output was the integral of many different processes. The discovery of Transport Inhibitor-Response 1 (TIR1) and the Auxin F-Box (AFB) proteins as sites of auxin perception and the role of auxin as molecular glue in the assembly of co-receptor complexes has allowed the development of a definitive quantitative structure-activity relationship for TIR1 and AFB5. Factorial analysis of binding activities offered two uncorrelated factors associated with binding efficiency and binding selectivity. The six maximum-likelihood estimators of Efficiency are changes in the overlap matrixes, inferring that Efficiency is related to the volume of the electronic system. Using the subset of compounds that bound strongly, chemometric analyses based on quantum chemical calculations and similarity and self-similarity indices yielded three classes of Specificity that relate to differential binding. Specificity may not be defined by any one specific atom or position and is influenced by coulomb matrixes, suggesting that it is driven by electrostatic forces. These analyses give the first receptor-specific classification of auxins and indicate that AFB5 is the preferred site for a number of auxinic herbicides by allowing interactions with analogues having van der Waals surfaces larger than that of indole-3-acetic acid. The quality factors are also examined in terms of long-standing models for the mechanism of auxin binding.


Assuntos
Proteínas de Arabidopsis/metabolismo , Proteínas F-Box/metabolismo , Ácidos Indolacéticos/metabolismo , Receptores de Superfície Celular/metabolismo , Proteínas Recombinantes de Fusão/metabolismo , Proteínas de Arabidopsis/química , Proteínas de Arabidopsis/genética , Baculoviridae/genética , Sítios de Ligação , Proteínas F-Box/química , Proteínas F-Box/genética , Vetores Genéticos , Ácidos Indolacéticos/química , Ligantes , Modelos Moleculares , Ligação Proteica , Receptores de Superfície Celular/química , Receptores de Superfície Celular/genética , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Relação Estrutura-Atividade , Ressonância de Plasmônio de Superfície
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