engleski

Expression and subcellular localization of human Nme6 protein in tumor cell lines

Nucleoside diphosphate kinase (NDPK/NME/Nm23) family is composed of enzymes catalyzing the transfer of gamma phosphate from nucleoside triphosphates to nucleoside diphosphates. The family has sparked considerable interest from the early nineties after the discovery of tumor metastasis regulation activity of NME1. The Group I members (NME1-NME4) are highly conserved in their amino acid sequence, highly homologous among themselves and exhibit NDPK activity, while Group II (NME5-NME9) members display less homology and seem to lack NDPK activity, with a possible exception of NME6. Although little is known about Group II members, those evolutionary very old genes are presumed to participate in one or more basic cellular process. Therefore, we focused our studies on revealing the subcellular localization, quaternary structure and function of the human Group II NME6 protein. The expression of NME6 was screened in several human tumor cell lines by Western blot, using specific anti-NME6 antibodies. Subcellular localization has been addressed using immunofluorescence coupled with confocal microscopy and confirmed by cell fractionation followed by Western blot analysis. Conventional cloning technics were used to obtain “knock-in” stable clones overexpressing NME6. CRISPR/Cas9 genome editing system was used for generating NME6 “knock-out” clones. All human tumor cell lines studied express significant amounts of NME6 protein. Immunofluorescence revealed the colocalization of NME6 predominantly with mitochondria. The cell fractionation confirmed the presence of NME6 in the mitochondrial fraction although human NME6 protein does not possess the mitochondrial targeting sequence. Complete NME6 “knock-out” clones could not be obtained, while partial NME6 “knock-outs” did not show decrease in NME6 expression, indicating that NME6 could plays a significant role in cell survival. Further studies will be conducted to detect the NME6 potential NDPK activity, interacting partners, its function in cellular processes and potential role in cancer.

Nme6 ; NDPK-H6

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