ИСТИНА

Caspase-2 fulfils pro- and nonapoptotic, as well as tumour-suppressor functions. This protein triggers apoptosis upon genotoxic stress and controls genomic stability, eliminating potentially dangerous cells. Nevertheless, the mechanisms of caspase-2 activation still remain unclear. For example, although caspase-2 was shown to be activated in PIDDosome complex, it could occur even in the absence of PIDD and/or RAIDD. Using mass spectrometry, caspase-2 has been found to interact with p62 and undergo ubiquitination. To clarify whether p62 regulates the caspase-2 autophagic or proteasomal degradation, these pathways were separately blocked. The inhibition of autophagy did not affect the caspase-2 level either under normal conditions or upon the genotoxic stress. Its level was restored only when the proteasome degradation was blocked upon the genotoxic stress. Pull-down assay revealed that caspase-2 underwent ubiquitination after DNA damage. Overexpression of p62 in cancer cells led to an accumulation of active caspase-2. The direct interaction between caspase-2 and p62 was confirmed via immunoprecipitation. Using BiFC method, we showed that mCherry-p62 was able to mediate the dimerization of caspase-2. Thus, p62 can act as an alternative platform for caspase-2 activation. To establish the role of ubiquitin in the interaction between caspase-2 and p62, the possibility of p62 without the ubiquitin-binding domain (p62-ΔUBD) to bind caspase-2 was analyzed. According to pull-down assay, p62-ΔUBD was not detected in caspase-2 samples, unlike the wild-type p62. However, using BiFC approach, mCherry-p62-ΔUBD was showed to promote dimerization and activation of caspase-2. Thus, the UBD of p62 is important for a stable binding with caspase-2. Importantly, p62 and caspase-2 can interact not only through ubiquitin, but also directly. Taken together, caspase-2 undergoes ubiquitination upon genotoxic stress and interacts with p62, which promotes its dimerization and activation. The levels of caspase-2 and p62 were also analyzed in tissue samples from lung adenocarcinoma patients. A statistically significant positive correlation was found between the levels of both proteins in tumour tissues. These data indicated the biological significance of the link between caspase-2 and p62.