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首页> 外文期刊>Biochimica et biophysica acta: BBA: International journal of biochemistry, biophysics and molecular biololgy. Proteins and Proteomics >Potential in vitro and ex vivo targeting of bZIP53 involved in stress response and seed maturation in Arabidopsis thaliana by five designed peptide inhibitors
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Potential in vitro and ex vivo targeting of bZIP53 involved in stress response and seed maturation in Arabidopsis thaliana by five designed peptide inhibitors

机译：Bzip53的体外和离体靶向参与拟南芥的应力反应和种子成熟，五种设计的肽抑制剂

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摘要

Basic leucine zipper (bZIP) transcription factors (TFs) are eukaryote-specific proteins that bind to DNA as a homodimer or heterodimer and regulate gene expression. They are involved in several biological processes in plants; therefore inhibiting bZIP-DNA binding activity by targeting protein-protein interface is an attractive proposition with aspects of both basic and applied biology. Here, we describe the equilibrium and kinetic interactions studies of a designed peptide inhibitor A-ZIP53 and its four variants with the bZIP53 protein, a key regulator of seed maturation phase and stress response in Arabidopsis. Five designed peptide inhibitors were primed to preferentially interact with bZIP53 and inhibit its DNA binding activity. Isothermal circular dichroism (CD) studies were used to quantify the structural changes accompanying heterodimers formation between bZIP53 and five A-ZIP53s. Equilibrium studies using electrophoretic mobility shift assay (EMSA) and fluorescence polarization (FP) assays suggest that A-ZIP53s and bZIP53 mixture form heterodimers, incapable of binding to DNA. Four A-ZIP53 derivatives were designed with additional interactions that drive heterodimerization with bZIP53. A-ZIP53s dose-dependent FP studies show that peptide inhibitors displaced the DNA bound bZIP53 with nM half-maximal inhibitory (IC50) concentrations. Using FP, time-dependent displacement kinetic studies were used to rank five A-ZIP53s for their abilities to displace DNA-bound bZIP53 with a rank order of A-ZIP53 A-ZIP53(A - E) A-ZIP53(N - A) A-ZIP53(R - E) A-ZIP53(A - E,N - A). In transient transfection assays, bZIP53-mediated GUS activity was inhibited by equimolar concentrations of five AZIP53s with A-ZIP53(A - E,N - A) the most effective one. Similar peptide inhibitors may be designed against other bZIP proteins to study their functions in vivo.
机译：基本亮氨酸拉链（BZIP）转录因子（TFS）是真核生物特异性蛋白质，其与DNA作为同型二聚体或异二聚体结合并调节基因表达。它们参与了植物中的几种生物过程;因此，通过靶向蛋白质 - 蛋白质界面抑制Bzip-DNA结合活性是一种具有基础和应用生物学的各方面的吸引力。这里，我们描述了设计的肽抑制剂A-ZIP53的平衡和动力学相互作用研究及其四种变体，其中具有BZIP53蛋白，种子成熟阶段的关键调节器和拟南芥的应力反应。将五种设计的肽抑制剂喷射以优先与BZIP53相互作用并抑制其DNA结合活性。等温圆形二色（CD）研究用于量化伴随BZIP53和5A-ZIP53s之间的异二聚体形成的结构变化。使用电泳迁移率移位测定（EMSA）和荧光极化（FP）测定的平衡研究表明A-ZIP53s和Bzip53混合物形成异二聚体，不能与DNA结合。四个A-ZIP53衍生物设计具有与Bzip53一起驱动异二聚体的额外相互作用。 A-Zip53S剂量依赖性FP研究表明，肽抑制剂用NM半最大抑制（IC50）浓度移位DNA结合的BZIP53。使用FP，时间依赖性位移动力学研究用于对5A-ZIP53进行排序，以使DNA结合的BZIP53与A-ZIP53的等级顺序排列的能力。 a-zip53（a - ＆ e）＆ A-ZIP53（n - ＆ a）＆ A-ZIP53（R - ＆ e）＆ A-ZIP53（a - ＆ e，n - ＆ a）。在瞬时转染测定中，BZIP53介导的GUS活性被5个Azip53s的等摩尔浓度抑制了53s（a - ＆ e，n - ＆ a）最有效的。类似的肽抑制剂可以针对其他Bzip蛋白设计，以研究其体内功能。

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