BACKGROUND: The formation of apoptosomes is well-established in the mechanism of programmed cell death. The interaction of APAF-1, caspase-3 and -9, which by adding of cytochrome C only in the presence of macroergs (dATP or ATP) generates apoptosome. Besides the cytoplasmic protein concentration is critical for the assembly of apoptosomes since in vitro it can induced only at values starting from ~2 mg/ml. The study of this feature led us to the discovery of at least one more protein that is critically involved in their formation.
METHODS: Cytoplasmic fraction from brain homogenates of newborn rats was obtained by centrifugation. Nucleoside di- and triphosphates, Na+, K+, RNAse A, DNAse1, phalloidin etc., as well as non-muscle F (filamentous) and G (monomeric) actin were added to study their effect on formation of active apoptosomes in the cytoplasm. Preliminary analysis showed that the proteasome activity of the "hydrolyzing peptidyl-glutamyl peptide" constitutes a significant part of all activities in the cleavage of caspase substrates. In this regard, experiments to study the activity of caspases were carried out in the presence of proteasome inhibitors (bortezomib or AdaAhx3L3VS), which do not affect the assembly of apoptosomes. Caspase activity was confirmed by the use of a selective caspase inhibitor emricasane.
RESULTS: It was found that manipulations with the cytoplasm (its concentration variation, the presence of monovalent cations, membrane fragments) in a dose-dependent manner nonlinearly led to an increasing of formed apoptosomes and the activity of caspase-3. These modifications changed the protease activity: maximal velocity from 0.15 to 2.4 mkmol/mg/min and Km from 4.2 to 0.13 mkM. A more detailed analysis showed that substances influencing on the assembly and disassembly of actin filaments directly affect both the formation of apoptosomes and the caspase-3 activity. This influence is critically significant, changing the activity by at least an order of magnitude. In this case, the effect of proteins that directly inhibit the activities of caspases (for example, XIAP) did not change.
CONCLUSIONS: Thus the actin G/F ratio (balance assembly/disruption of actin filaments) is a key regulator of the assembly of apoptosomes and it depends on the presence of nucleoside triphosphates. It explains the critical dependence of apoptosis on the production of macroergs.
