ИСТИНА

Caspase-2 was the first identified human caspase and still remains the least studied. Сaspase-2, as a member of the caspase family, fulfills both pro-apoptotic and tumor-suppressive functions. This protein controls maintenance of genomic stability and triggers apoptosis in response to genotoxic stress. Recent studies have demonstrated its role in the elimination of potentially dangerous oncogenic aneuploid cells, resulting from a disruption of the cell division process. However, the detailed mechanisms for the activation and functioning of caspase-2 remain unclear and require further investigation. Post-translational modifications (PTMs), e.g. phosphorylation and ubiquitination, are known to play a crucial role in regulation of caspases activation. Although, multiple PTMs have been reported for initiator caspase-8 and -9, only three cases of phosphorylation have been described for caspase-2. Bioinformatic analysis allowed to predict potential phosphorylation sites of caspase-2 - S196, S220, S307, S384 and T380, which could regulate caspase-2 activation. The mutant variants of caspase-2 with substitutions S196D, S220A, S307A, S384A and T380A were generated. Wild-type caspase-2 and the four mutated variants of caspase-2 with substitutions S196D, S220A, S307A, and T380A have been shown to undergo autocatalytic activation that led to accumulation of p32 and p19 active fragments. The mutation S384A was able to block caspase-2 processing. Moreover, genotoxic stress enhanced accumulation of caspase-2 active fragments; however, the substitution S384A fully inhibited cisplatin-induced generation of active subunits. Additionally, isolation of caspase-2 using affinity chromatography has shown that caspase-2S384A did not contain the p19 active fragment. Consequently, these data confirmed that the mutation S384A in caspase-2 led to full inhibition of autocatalytic activation. Measurement of caspase-2 activity using fluorogenic peptide Ac-VDVAD-AMC corroborated that caspase-2S384A did not have catalytic activity under genotoxic stress. Taken together, the mutation of potential phosphorylation site S384 caused not only blockage of caspase-2 processing but also inhibition of its catalytic activity. Additionally, caspase-2 has been found to undergo ubiquitination. Under genotoxic stress caspase-2 was enriched with K48-linked polyubiquitin chains that suggested enhancement of its proteasomal degradation. MG132-mediated inhibition of proteasomal degradation led to accumulation of caspase-2 p19 fragment at 4 and 8 hours of incubation. The accumulation of p19 was enhanced under combined treatment with MG-132 and DNA-damaging agents. A decrease of procaspase-2 level was not detected, suggesting that the accumulation of p19 subunit was caused by inhibition of its proteasomal degradation but not through enchantment of procaspase-2 processing. Thus, under genotoxic stress caspase-2, in its active cleaved forms, undergoes ubiquitination that leads to its degradation. An inhibition of proteasomal degradation results in accumulation of active caspase-2 that stimulates cell death.