engleski

Characterization of ancestral-type Grope I Nm23 gene/protein - structure and function is conserved from sponges to humans

Title: Characterization of ancestral-type Group I Nm23 gene/protein - structure and function is conserved from sponges to humans Authors: Dragutin Perina, Maja Herak Bosnar, Ruzica Bago, Andreja Mikoc, Matija Harcet, Martina Dezeljin, Helena Cetkovic Aims: The goal of this study was to determine the gene structure and study the biochemical characteristics and functions of the ancestral-type sponge protein Nm23-SD1. Furthermore, we wanted to test whether it can substitute the most recent (and the most studied) human Group I member –Nm23-H1 and explore if the sponge homologue has the ability to suppress the metastatic potential of human cancer cells in vitro. Hypothesis: According to nucleotide and aminoacid sequence homology our hypothesis was that the sponge Nm23-SD1 has biochemical characteristics similar to human Nm23-H1/H2 gene/protein, and therefore should behave similar to them if introduced into human cancer cells. Methods: Multiple sequence alignments (MSA) and construction of the phylogenetic tree from the MSA were performed with CLUSTALX and MEGA4. For biochemical studies the recombinant protein was purified and its structure determined by gel filtration and glutaraldehyde crosslinking assay. The Nm23-SD1 activity was revealed by standard NDPK assay, and further characterized by ssDNA binding assay, and plasmid cleavage assay. For subcellular localization studies the Nm23-SD1 and Nm23-H1 were subcloned to pEGFP or pDsRed reporter systems, transfected into HEp-2 cells, and analyzed using confocal laser scanning microscope. The migration assays was preformed on CAL 27 cell line stable clones expressing FLAG/Nm23-SD1 compared to FLAG/Nm23-H1 overexpressing CAL 27 clones. The FLAG/Nm23-H1 and FLAG/Nm23-SD1 complex formation was determined by anti FLAG M2 affinity gel and Western blotting. Results: The ancestral metazoan Group I nm23 gene was intron-rich and probably had four introns with three out of four well preserved in vertebrate homologues. Nm23-SD1 protein has a similar level of kinase activity as its human homolog, does not cleave negatively supercoiled DNA and shows nonspecific DNA-binding activity. The sponge Nm23-SD1 forms a hexamer, like all other eukaryotic Nm23 proteins. Nm23-SD1 interacts with its human homolog Nm23-H1 in human cells and exhibits the same subcellular localization. Stable clones expressing sponge Nm23-SD1 inhibited the migratory potential of CAL 27 cells, as already reported for Nm23-H1. Conclusions: Based on the evidence we collected we can conclude: i) sponge nm23 gene and Nm23 protein reflects the characteristics of the ancestral Group I nm23 gene/protein ; ii) Nm23-SD1 possesses many biochemical properties of the most recent Group I variant, the Nm23-H1 homolog ; iii) Nm23- SD1 interacts with Nm23-H1 and possibly replaces it in some biological functions which are usually associated with higher metazoans. References: Harcet M, et al. Croat Chem Acta 2005, 78(3):343- 348. Bago R, et al. Mol Carcinogen 2009, 48(9):779-789.

nm23; NDPK; sponges; ancestral gene

nije evidentirano