INVESTIGADORES
AGÜERO Tristan Horacio
artículos
Título:
Dissecting CNBP, a zinc-finger protein required for neural crest development, in its structural and functional domains
Autor/es:
PABLO ARMAS; TRISTÁN H. AGÜERO; MARIANA BORGOGNONE; MANUEL J. AYBAR; NORA B. CALCATERRA
Revista:
JOURNAL OF MOLECULAR BIOLOGY
Editorial:
ELSEVIER
Referencias:
Año: 2008 p. 1043 - 1056
ISSN:
0022-2836
Resumen:
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 acid and displays nucleic acid chaperone activity in vitro. CNBP family shows a conserved modular organization of seven Zn knuckles and an 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. The RGG box removal allowed CNBP to preserve a weak ssDNA binding capability. A mutant mimicking the natural N-terminal proteolytic CNBP form behaved as the RGG deleted mutant. By gain- 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.