Caspase-2 engages the mitochondria-dependent apoptotic pathway by inducing the release of cytochrome c. Previous studies in Xenopus laevis oocytes have identified that phosphorylation of procaspase-2 (proC2) at several residues under nutrient abundance conditions suppresses its activation and subsequently blocks the apoptosis through process involving the binding of the scaffolding protein 14-3-3 [1]. To elucidate the mechanism of the 14-3-3 protein-mediated inhibition of caspase-2 in human, we used various biochemical and biophysical approaches to identify phosphorylated motifs of human proC2 required for 14-3-3 binding. Using fluorescence polarization assay, native electrophoresis and analytical gel filtration we show that the 14-3-3:proC2 complex assembling is governed by both of these binding sites: S139 and S164 [2].
Characterization of proC2 in solution showed its monomeric status (methods of analytical ultracentrifugation and small-angle X-ray scattering). By continuing studies of the 14-3-3:proC2 complex we determined that the dissociation constant between 14-3-3:proC2 is in a nanomolar range and the stoichiometry of the interaction is 2:1. Data also indicate that other regions of proC2, in addition to phosphorylation motifs, may be involved in the interaction with 14-3-3. Limited proteolysis of proC2 alone and in the complex with 14-3-3 protein by trypsin and chymotrypsin found out the capability of 14-3-3 to protect the proC2 N-terminal part against proteolytic degradation. The circular dichroism spectra measurements detected significant changes in the tertiary structure of proC2 after 14-3-3:proC2 complex formation.
This study was supported by the Czech Science Foundation (Projects 17-00726S), the Czech Academy of Sciences (Research Projects RVO: 67985823 of the Institute of Physiology).