| Popis: |
The Nme gene/protein family of nucleoside diphosphate kinases (NDPK), was originally named after its member Nm23-H1/Nme1. It was the first gene identified as metastasis suppressor. To date, ten different Nme genes have been identified in humans, with Nme1 and Nme2 as the most studied representatives. Nme proteins in humans have been divided in Group I (Nme1-Nme4) and Group II (Nme5-Nme9) and all of them possess nucleoside diphosphate kinase domain (NDK). There is a single histidine residue involved in the catalytic mechanism, conserved in all known active NDPK enzymes. Group I proteins display a single type NDK domain, whereas Group II proteins display a single or several NDK domains of different types, associated or not with extra-domains. In contrast to Group I members, the accumulating data strongly suggest that none of the Group II members display measurable NDPK activities, even though some of them are able to autophosphorylate. Multimeric form of any Group II member is not determined yet. Our comprehensive analyses of the Nme family in eukaryotes revealed conservancy of ancestral type of Group II protein in several species from three eukaryotic supergroups. Therefore we analyzed Nme protein from early branching eukaryotic lineage. We chose Chondrus crispus (Irish moss), recently sequenced red alga which is an excellent model organism for understanding Nme evolution. Genomic DNA from C. crispus was isolated and plasmids for overproduction of recombinant protein, localization assay, cell proliferation, apoptosis and soft agar colonization assay were constructed. NDPK activity was measured using a coupled PK-LDH (pyruvate kinase-lactate dehydrogenase) assay and the fast protein liquid chromatography (FPLC) system was employed for determination of protein MW. HeLa and HEK293T cells were used for transfection. Our analyses revealed that ancestral type of protein, in contrast to human homologue, was fully functional multimeric NDP kinase. This three-domain compound protein shows high affinity to various types of DNA and displays a dispersed localization throughout the eukaryotic cell. Overexpression of enzyme inhibits both cell proliferation and anchorage independent growth in soft agar, but does not deregulate the cell apoptosis. We conclude that the structure of ancestral gene has changed during eukaryotic evolution possibly in correlation with the function of the protein. |
