The process of programmed cell death, apoptosis, is generally characterized by distinct morphological characteristics and energy-dependent biochemical mechanisms. Apoptosis is considered a vital component of various processes including normal cell turnover, proper development and functioning of the immune system, hormone-dependent atrophy, embryonic development and chemical-induced cell death. Caspases were identified to play a crucial role in apoptotic pathways. It was described previously that sufficient NADPH level induces phosphorylation of procaspase-2 (proC2) and 14-3-3 protein binding preventing proC2 maturation. The nutrient depletion promotes the 14-3-3 protein release and caspase-2 activation as was described previously in Xenopus laevis. [1] Our recent research suggested that two phosphorylation sites are required for proC2 binding to 14-3-3 [2].
Conformational behavior of phosphorylated proC2 and its changes upon 14-3-3 protein binding was studied next by time resolved fluorescence intensity and decay measurements and acrylamide quenching. Four proC2 mutants containing single tryptophan residue at positions 151, 188, 218 and 426 and wt (containing Trp385), were prepared to sample various regions of proC2. Values of mean fluorescence lifetimes clearly show the different vicinity in individual mutants after 14-3-3 binding with exception of Trp188 which seems to be buried within the structure of proC2. Data obtained from fluorescence anisotropy determined Trp151 and Trp426 exhibit significantly lower fast local motions of Trp upon complex formation, indicating lower mobility of this region upon 14-3-3 protein binding.
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).