engleski

Identification, expression and subcellular localization of human NME6 protein

Nucleoside diphosphate kinase (NDPK/NME/Nm23) family is divided in two groups: Group I (NME1- NME4) members are highly homologous among themselves and exhibit NDPK activity ; Group II (NME5-NME9) members display less homology and seem to lack NDPK activity, with a possible exception of NME6 which contains the eleven amino acids important for functional kinase activity. Extensive research has been conducted on attractive Group I members, while Group II did not raise the same interest. Although little is known about Group II members, these evolutionary very old genes are presumed to participate in one or more basic cellular process. Therefore, we focused our studies on Group II human NME6 protein, to identify endogenous NME6 isoforms, monitor their expression, and reveal their subcellular localization, quaternary structure and function. Identification of endogenous NME6 was achieved by mass spectrometry (LC-MS) along with Western Blot analysis comparing endogenous protein and recombinant isoforms. The expression of NME6 in human cancer cell lines was screened by Western Blot using specific anti-NME6 antibodies. Subcellular localization was investigated by immunofluorescence coupled with confocal microscopy and consolidated by cell fractionation followed by Western Blot analysis. To understand the NME6 function, conventional techniques were used to obtain “knock-in” stable clones overexpressing exogenous human NME6- FLAG. CRISPR/Cas9 genome editing system was used to generate stable NME6 “knock-outs”. The main human endogenous NME6 protein expressed seem to be the isoform of 186aa, although the longer isoform (194aa) was also detected. All human cancer cell lines studied express significant and similar amounts of NME6 protein. Immunofluorescence revealed the colocalization of NME6 predominantly with mitochondria. Cell fractionation confirmed the presence of NME6 in the mitochondrial fraction although human NME6 protein does not possess the mitochondrial targeting sequence. Overexpressed exogenous NME6-FLAG proteins from “knock-in” also colocalizes with mitochondria. Only monoallelic NME6 “knock-out” clones were produced, likely indicating that NME6 plays a significant role in cell survival. Further studies will be conducted to detect the NME6 potential NDPK activity, quaternary structure, its function in cellular processes and potential role in cancer.

NME6 ; localization ; mitochondria

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