Armas Pablo, Agüero Tristán H, Borgognone Mariana, Aybar Manuel J, Calcaterra Nora B
División Biología del Desarrollo, Instituto de Biología Molecular y Celular de Rosario, Facultad de Ciencias Bioquímicas y Farmacéuticas, Universidad Nacional de Rosario, Suipacha 531, S2002LRK Rosario, Argentina.
J Mol Biol. 2008 Oct 17;382(4):1043-56. doi: 10.1016/j.jmb.2008.07.079. Epub 2008 Aug 5.
Cellular nucleic-acid-binding protein (CNBP) plays an essential role in forebrain and craniofacial development by controlling cell proliferation and survival to mediate neural crest expansion. CNBP binds to single-stranded nucleic acids and displays nucleic acid chaperone activity in vitro. The CNBP family shows a conserved modular organization of seven Zn knuckles and an arginine-glycine-glycine (RGG) box between the first and second Zn knuckles. The participation of these structural motifs in CNBP biochemical activities has still not been addressed. Here, we describe the generation of CNBP mutants that dissect the protein into regions with structurally and functionally distinct properties. Mutagenesis approaches were followed to generate: (i) an amino acid replacement that disrupted the fifth Zn knuckle; (ii) N-terminal deletions that removed the first Zn knuckle and the RGG box, or the RGG box alone; and (iii) a C-terminal deletion that eliminated the three last Zn knuckles. Mutant proteins were overexpressed in Escherichia coli, purified, and used to analyze their biochemical features in vitro, or overexpressed in Xenopus laevis embryos to study their function in vivo during neural crest cell development. We found that the Zn knuckles are required, but not individually essential, for CNBP biochemical activities, whereas the RGG box is essential for RNA-protein binding and nucleic acid chaperone activity. Removal of the RGG box allowed CNBP to preserve a weak single-stranded-DNA-binding capability. A mutant mimicking the natural N-terminal proteolytic CNBP form behaved as the RGG-deleted mutant. By gain-of-function and loss-of-function experiments in Xenopus embryos, we confirmed the participation of CNBP in neural crest development, and we demonstrated that the CNBP mutants lacking the N-terminal region or the RGG box alone may act as dominant negatives in vivo. Based on these data, we speculate about the existence of a specific proteolytic mechanism for the regulation of CNBP biochemical activities during neural crest development.
细胞核酸结合蛋白（CNBP）通过控制细胞增殖和存活来介导神经嵴扩展，在前脑和颅面发育中发挥着至关重要的作用。CNBP与单链核酸结合，并在体外表现出核酸伴侣活性。CNBP家族显示出由七个锌指和第一个与第二个锌指之间的精氨酸 - 甘氨酸 - 甘氨酸（RGG）框组成的保守模块化结构。这些结构基序在CNBP生化活性中的参与情况尚未得到研究。在此，我们描述了CNBP突变体的产生，这些突变体将蛋白质分解为具有结构和功能不同特性的区域。采用诱变方法产生：（i）破坏第五个锌指的氨基酸替换；（ii）去除第一个锌指和RGG框或仅去除RGG框的N端缺失；（iii）去除最后三个锌指的C端缺失。突变蛋白在大肠杆菌中过表达、纯化，并用于体外分析其生化特性，或在非洲爪蟾胚胎中过表达以研究其在神经嵴细胞发育过程中的体内功能。我们发现锌指对于CNBP生化活性是必需的，但并非单个锌指必不可少，而RGG框对于RNA - 蛋白质结合和核酸伴侣活性至关重要。去除RGG框使CNBP保留了较弱的单链DNA结合能力。模拟天然N端蛋白水解CNBP形式的突变体表现得与缺失RGG框的突变体一样。通过在非洲爪蟾胚胎中的功能获得和功能丧失实验，我们证实了CNBP参与神经嵴发育，并且证明了单独缺失N端区域或RGG框的CNBP突变体在体内可能作为显性负性因子起作用。基于这些数据，我们推测在神经嵴发育过程中存在一种特定的蛋白水解机制来调节CNBP的生化活性。
