ИСТИНА

NRH:quinoneoxidoreductase 2 (NQO2) is a cytosolic protein that catalyzes metabolism of quinones. NQO2 is ubiquitously present in all tissues and induced along with a battery of defensive genes in response to different stresses. It is concerned that NQO2 plays an important role in development of neurodegenerative disorders and cancer, but the mechanism of its action in disease initiation and progression is unknown. The same is definitely true for p53 and mitochondria being the most important regulatory protein and organelle in human cells and playing crucial roles in cancer, neurodegenaration, inflammation and ageing. Perhaps, NQO2, p53, and mitochondria could act in cooperation. We previously found that NQO2 contributes to the p53 stabilization and activation after mitochondrial electron transport chain complex III inhibition. But the mechanism of its action is unknown. To study the possible interaction of NQO2 and p53 in different cancer cell lines under normal conditions and after mitochondrial electron transport chain inhibition we used Bimolecular Fluorescence Complementation (BiFC) method. N-terminal (N-YFP) and C-terminal (C-YFP) fragments of YFP were fused to p53 and NQO2 proteins and the fusion proteins were expressed in HeLa or RKO cells. Expression of fusion proteins was verified by Westernblot analysis with NQO2 and p53 antibody. Recovery of the whole YFP fluorescence was detected in the cells expressing C-YFPNQO2 and N-YFP-p53 by fluorescent microscopy (picture below). Thus, using BiFC we received rather strong experimental evidence that p53 forms a complex with NQO2 in cells under normal conditions and after mitochondrial complex III inhibition. According to immunofluorescent images under normal conditions NQO2-p53 complexes are localized predominantly in the cytoplasm, and after complex III inhibition they can be seen mainly in cell nuclei, where p53 performs its function. This result is a good start for investigation of the role and detailed mechanism of p53-NQO2 interaction in cancer cells under normal conditions and mitochondrial dysfunction.