engleski

Expression and subcellular localization of Nme6- Nm23-H6

NME6 is a member of the nucleoside diphosphate kinase (NDPK/NME/Nm23) family, enzymes that catalyze the transfer of gamma phosphate from ATP to other nucleoside diphosphates. The family has attracted considerable interest due to the involvement of its members in metastasis suppression. The family is divided into two groups. Group I (NME1-NME4) are highly conserved in their amino acid sequence and exhibit NDPK activity, while Group II (NME5- NME9) members display less homology and seem to lack the NDPK activity, with a possible exception of NME6. Some of the Group II members (NME5-7) arose very early in the evolution of Metazoa, therefore, it is presumed that they participate in one or more basic cellular process. Since little is known about Group II members, we focused our studies on revealing the subcellular localization, quaternary structure and function of Group II human 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 confirmed by cell fractionation followed by Western blot analysis. Immunofluorescence assays coupled with confocal microscopy has been used to validate results. All human tumor cell lines studied express significant amounts of NME6 protein. Immunofluorescence revealed the colocalization of NME6 with mitochondria which was confirmed by cell fractionation although human NME6 protein does not possess the mitochondrial targeting sequence. We have produced monoalelic CRISPR/Cas9 knock-outs and produced two recombinant protein isoforms. Further studies will be conducted to detect the NME6 potential NDPK activity, quaternary structure, its function in mitochondrial respiration and potential role in cancer.

Nm23-H6 ; NME6 ; NDPK ; subcellular localization

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