RESUMO
Tobacco cytochrome P450 (CYP) 71AH11 metabolized the herbicide chlorotoluron, and its mRNA level was increased in tobacco culture cells by the treatment of 2,4-D. In order to clarify molecular mechanisms of induced gene expression of CYP71AH11 by herbicide treatment, a 1574-bp 5'-flanking region of CYP71AH11 was cloned, ligated to the reporter ß-glucuronidase (GUS) gene, and then transformed into tobacco plants. The GUS activity in the transgenic tobacco plants was highly induced by bromoxynil treatment, followed by 2,4-D. Chlorotoluron was slightly increased the GUS activity. The bromoxynil-increased GUS activity was partially repressed by the antioxidants, suggesting that reactive oxygen species may be involved in activation of the 5'-flanking region of CYP71AH11 by bromoxynil treatment. Deletion and mutation assays showed that the region CD (-1281 to -770bp from the start codon of CYP71AH11) was important, but not sufficient, for response to bromoxynil. Electrophoretic mobility shift assays and southwestern blotting revealed that the sequences AAAAAG, and GAACAAAC and GAAAATTC in the CD region were important for interaction to the nuclear proteins of <30 and ≈75 kDa, respectively. Particularly, interaction between AAAAAG and <30 kDa proteins was increased by bromoxynil treatment. These results gave a cue for understanding the bromoxynil-induced gene expression of CYP71AH11, which may contribute to herbicide tolerance and selectivity in crop plants.
Assuntos
Sistema Enzimático do Citocromo P-450/metabolismo , Regulação Enzimológica da Expressão Gênica , Herbicidas/metabolismo , Nicotiana/enzimologia , Compostos de Fenilureia/metabolismo , Proteínas de Plantas/metabolismo , Sequência de Bases , Sistema Enzimático do Citocromo P-450/genética , Regulação da Expressão Gênica de Plantas , Dados de Sequência Molecular , Proteínas de Plantas/genética , Regiões Promotoras Genéticas , Nicotiana/genética , Nicotiana/metabolismoRESUMO
When a large hydrophobic full-length protein is expressed in bacteria, it is often challenging to obtain recombinant proteins in the soluble fraction. One way to overcome this challenge is expression of deletion mutants that have improved solubility while maintaining biological activity. In this chapter, we describe a protocol for expression of truncated forms of STAT5b and STAT3 proteins that are soluble and retain SH2-mediated activity for phospho-Tyr peptide recognition.
Assuntos
Expressão Gênica , Proteínas Recombinantes , Fator de Transcrição STAT3/genética , Fator de Transcrição STAT3/isolamento & purificação , Fator de Transcrição STAT5/genética , Fator de Transcrição STAT5/isolamento & purificação , Bioensaio , Humanos , Plasmídeos/genética , Ligação Proteica , Fator de Transcrição STAT3/química , Fator de Transcrição STAT3/metabolismo , Fator de Transcrição STAT5/química , Fator de Transcrição STAT5/metabolismo , Solubilidade , Domínios de Homologia de srcRESUMO
Constitutive activation of STAT3/5b frequently occurs in various human malignancies. STAT3/5b activation involves dimerization via intermolecular pTyr-SH2 binding; therefore, antagonizing this interaction is a feasible approach to inhibit STAT3/5b activation for cancer therapy. We have developed a multiplexed assay to assess STAT3- and STAT5b-SH2 binding in a single well by combining AlphaLISA and AlphaScreen beads. In this chapter, we describe application of the method for the purpose of identifying new STAT3 and STAT5b antagonists.
Assuntos
Bioensaio , Descoberta de Drogas , Proteínas/química , Domínios de Homologia de src , Bioensaio/métodos , Relação Dose-Resposta a Droga , Descoberta de Drogas/métodos , Ensaios de Triagem em Larga Escala , Humanos , Ligantes , Ligação Proteica , Proteínas/antagonistas & inibidores , Fator de Transcrição STAT3/antagonistas & inibidores , Fator de Transcrição STAT3/química , Fator de Transcrição STAT3/metabolismo , Fator de Transcrição STAT5/antagonistas & inibidores , Fator de Transcrição STAT5/química , Fator de Transcrição STAT5/metabolismo , Bibliotecas de Moléculas PequenasRESUMO
Some of the signal transducer and activator of transcription (STAT) family members are constitutively activated in a wide variety of human tumors. The activity of STAT depends on their Src homology 2 (SH2) domain-mediated binding to sequences containing phosphorylated tyrosine. Thus, antagonizing this binding is a feasible approach to inhibiting STAT activation. We have developed a novel multiplexed assay for STAT3- and STAT5b-SH2 binding, based on amplified luminescent proximity homogeneous assay (Alpha) technology. AlphaLISA and AlphaScreen beads were combined in a single-well assay, which allowed the binding of STAT3- and STAT5b-SH2 to phosphotyrosine peptides to be simultaneously monitored. Biotin-labeled recombinant human STAT proteins were obtained as N- and C-terminal deletion mutants. The spacer length of the DIG-labeled peptide, the reaction time, and the concentration of sodium chloride were optimized to establish a HTS system with Z' values of greater than 0.6 for both STAT3- and STAT5b-SH2 binding. We performed a HTS campaign for chemical libraries using this multiplexed assay and identified hit compounds. A 2-chloro-1,4-naphthalenedione derivative, Compound 1, preferentially inhibited STAT3-SH2 binding in vitro, and the nuclear translocation of STAT3 in HeLa cells. Initial structure activity relationship (SAR) studies using the multiplexed assay showed the 3-substituent effect on both the activity and selectivity of STAT3 and STAT5b inhibition. Therefore, this multiplexed assay is useful for not only searching for potential lead compounds but also obtaining SAR data for developing new STAT3/STAT5b inhibitors.