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.
© 2024. The Author(s).

This study reports a dose-dependent pro-apoptotic action of synthetic cannabimimetic N-stearoylethanolamine (NSE) on diverse cancer cell lines, including multidrug-resistant models. No antioxidant or cytoprotective effects of NSE were found when it was applied together with doxorubicin. A complex of NSE with the polymeric carrier poly(5-(tert-butylperoxy)-5-methyl-1-hexen-3-yn-co-glycidyl methacrylate)-graft-PEG was synthesized. Co-immobilization of NSE and doxorubicin on this carrier led to a 2-10-fold enhancement of the anticancer activity, particularly, against drug-resistant cells overexpressing ABCC1 and ABCB1. This effect might be caused by accelerated nuclear accumulation of doxorubicin in cancer cells, which led to the activation of the caspase cascade, revealed by Western blot analysis. The NSE-containing polymeric carrier was also able to significantly enhance the therapeutic activity of doxorubicin in mice with implanted NK/Ly lymphoma or L1210 leukemia, leading to the complete eradication of these malignancies. Simultaneously, loading to the carrier prevented doxorubicin-induced elevation of AST and ALT as well as leukopenia in healthy Balb/c mice. Thus, a unique bi-functionality of the novel pharmaceutical formulation of NSE was revealed. It enhanced doxorubicin-induced apoptosis in cancer cells in vitro and promoted its anticancer activity against lymphoma and leukemia models in vivo. Simultaneously, it was very well tolerated preventing frequently observed doxorubicin-associated adverse effects.

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.

Colorectal cancer (CRC) is one of the most common cancers that is characterized by a high mortality due to the strong metastatic potential of the primary tumor and the high rate of therapy resistance. Hereby, evasion of apoptosis is the primary underlying cause of reduced sensitivity of tumor cells to chemo- and radiotherapy. Using RNA affinity chromatography, we identified the tripartite motif-containing protein 25 (TRIM25) as a bona fide caspase-2 mRNA-binding protein in colon carcinoma cells. Loss-of-function and gain-of-function approaches revealed that TRIM25 attenuates the protein levels of caspase-2 without significantly affecting caspase-2 mRNA levels. In addition, experiments with cycloheximide revealed that TRIM25 does not affect the protein stability of caspase-2. Furthermore, silencing of TRIM25 induced a significant redistribution of caspase-2 transcripts from RNP particles to translational active polysomes, indicating that TRIM25 negatively interferes with caspase-2 translation. Functionally, the elevation in caspase-2 upon TRIM25 depletion significantly increased the sensitivity of colorectal cells to drug-induced intrinsic apoptosis as implicated by increased caspase-3 cleavage and cytochrome c release. Importantly, the apoptosis-sensitizing effects by transient TRIM25 knockdown were rescued by concomitant silencing of caspase-2, demonstrating a critical role of caspase-2. Inhibition of caspase-2 by TRIM25 implies a survival mechanism that critically contributes to chemotherapeutic drug resistance in CRC.

Inflammatory caspases (caspase-1, caspase-4, caspase-5 and caspase-11 (caspase-1/-4/-5/-11)) mediate host defense against microbial infections, processing pro-inflammatory cytokines and triggering pyroptosis. However, precise checkpoints are required to prevent their unsolicited activation. Here we report that serpin family B member 1 (SERPINB1) limited the activity of those caspases by suppressing their caspase-recruitment domain (CARD) oligomerization and enzymatic activation. While the reactive center loop of SERPINB1 inhibits neutrophil serine proteases, its carboxy-terminal CARD-binding motif restrained the activation of pro-caspase-1/-4/-5/-11. Consequently, knockdown or deletion of SERPINB1 prompted spontaneous activation of caspase-1/-4/-5/-11, release of the cytokine IL-1β and pyroptosis, inducing elevated inflammation after non-hygienic co-housing with pet-store mice and enhanced sensitivity to lipopolysaccharide- or Acinetobacter baumannii-induced endotoxemia. Our results reveal that SERPINB1 acts as a vital gatekeeper of inflammation by restraining neutrophil serine proteases and inflammatory caspases in a genetically and functionally separable manner.