Mitotic catastrophe is a defensive mechanism that promotes elimination of cells with aberrant mitosis by triggering the cell-death pathways and/or cellular senescence. Nowadays, it is known that apoptosis, autophagic cell death, and necrosis could be consequences of mitotic catastrophe. Here, we demonstrate the ability of a DNA-damaging agent, doxorubicin, at 600 nM concentration to stimulate mitotic catastrophe. We observe that the inhibition of caspase activity leads to accumulation of cells with mitotic catastrophe hallmarks in which RIP1-dependent necroptotic cell death is triggered. The suppression of autophagy by a chemical inhibitor or ATG13 knockout upregulates RIP1 phosphorylation and promotes necroptotic cell death. Thus, in certain conditions mitotic catastrophe, in addition to apoptosis and autophagy, can precede necroptosis.

Caspase-2 is a unique and conservative cysteine protease which plays an important role in several cellular processes including apoptotic cell death. Although the molecular mechanisms of its activation remain largely unclear, a major role belongs to the architecture of the caspase-2 active center. We demonstrate that the substitution of the putative phosphorylation site of caspase-2, Serine-384 to Alanine, blocks caspase-2 processing and decreases its enzymatic activity. Strikingly, in silico analysis using molecular dynamics simulations has shown that Serine-384 is crucially involved in interactions within the caspase-2 active center. It stabilizes Arginine-378, which forms a crucial hydrogen bond with the aspartate residue of a substrate. Hence, Serine-384 is essential for supporting a proper architecture of the active center of caspase-2. Moreover, molecular modeling strongly proved steric inaccessibility of Ser-384 to be phosphorylated. Importantly, a multiple alignment has demonstrated that both Serine-384 and Arg-378 residues are highly conservative across all members of caspase family, which allows us to suggest that this diade is indispensable for caspase processing and activity. Spontaneous mutations in this diade might influence oncosuppressive function of caspases, in particular of caspase-2. Likewise, the mutation of Ser-384 is associated with the development of lung squamous cell carcinoma and adenocarcinoma. Taken together, we have uncovered a central feature of the caspase-2 activation mechanism which is crucial for the regulation of its signaling network.

Caspase-2 is a unique and conserved cysteine protease that is involved in several cellular processes, including different forms of cell death, maintenance of genomic stability, and the response to reactive oxygen species. Despite advances in caspase-2 research in recent years, the mechanisms underlying its activation remain largely unclear. Although caspase-2 is activated in the PIDDosome complex, its processing could occur even in the absence of PIDD1 and/or RAIDD, suggesting the existence of an alternative platform for caspase-2 activation. Here, we show that caspase-2 undergoes ubiquitination and interacts with scaffolding protein p62/sequestosome-1 (SQSTM1) under normal conditions and in response to DNA damage. p62 promotes proteasomal but not autophagic caspase-2 degradation as well as its dimerization and activation that triggers the caspase cascade and, subsequently, cell death. Inhibition of p62 expression attenuates cisplatin-induced caspase-2 processing and apoptosis. Notably, the ZZ domain of p62 is critical for caspase-2 binding, whereas the UBA domain is seemingly required to stabilize the p62–caspase-2 complex. Thus, we have uncovered the dual role of p62 in regulating caspase-2 activity: it can foster the degradation of caspase-2 in the proteasome or facilitate its activation by acting as a scaffold platform.

Rol' kaspazy-2 v gibeli kletok kartsinomy yaichnika po puti mitoticheskoy katastrofy Роль каспазы-2 в гибели клеток карциномы яичника по пути митотической катастрофы

An aptamer’s low mass confines the label-free response to hundredths of a nanometer, where artifacts mimic binding yet yield computable dissociation constants. To define and test a priori criteria distinguishing binding from artifact, 88 computationally designed aptamers against seven human proteins were studied by biolayer interferometry in two orthogonal configurations, with control antibodies. Six criteria, fixed in advance, are reported as a priori decision rules, not validated requirements. Control antibodies validated 6 of 8 preparations; for the other 68 pairs, the maximum response was 0.019–0.219 nm, bounding K_D at ≥8.6 µM, 120-fold above the 70 nM threshold. Two of three signals clearing it gave exponents far above the 1:1 expectation (1.04 vs. 0.31; 0.85 vs. 0.41); the third failed parameter consistency (K_D scatter ×28). Rankings on two PCSK9 preparations did not correlate (ρ = −0.26); predicted and experimental K_D differed by 3–7 orders of magnitude (ρ = 0.497, n = 25). No aptamer reached the predefined affinity under the conditions tested. Local fitting returns internally consistent K_D for data incompatible with a 1:1 model; we propose isotherm verification and antibody validation of every preparation as minimal requirements. No positive-control aptamer was available, limiting interpretation.