Abstract
Apoptosis is a process of programmed cell death that maintain number of cells in tissues. This set of biochemical pathways occurs for instance during aging or immune reactions.
Caspases were identified to play a crucial role in apoptotic pathways and they are named due to their specific cysteine protease activity. Our research is focused on human proCaspase-2 and its interaction with the 14-3-3 protein which was described in Xenopus laevis [1].
The sufficient NADPH level induces phosphorylation of Caspase-2 and 14-3-3 protein binding prevents proCaspase-2 maturation and as a result inhibiting the apoptosis. The nutrient depletion promotes the 14-3-3 protein release and Caspase-2 activation.
ProCaspase-2 as an inactive proenzyme consists of 3 domains: CARD, p18 and p12. CARD and p18 are connected with linker containing phosphorylated Ser 139 and Ser 164 [2].
Our recent research confirmed the phosphorylation dependent interaction of proCaspase-2 with the 14-3-3 protein. In current study we tested conformational behavior of phosphorylated proCaspase-2 and its changes upon complex formation with 14-3-3 using time resolved fluorescence measurements.
Four procaspase-2 mutants and WT containing single tryptophan residue were prepared at positions 151, 188, 218 and 426, to sample various regions of proCaspase-2. Values of mean fluorescence lifetimes (τ mean ) clearly show the different vicinity in individual mutants after the 14-3-3 binding with exception of Trp 188 which seems to be buried within the structure of proCaspase-2.
Data obtained from fluorescence anisotropy determined Trp 151 and Trp 426 with flexible vicinity with significant restriction after 14-3-3 binding. On the other hand area around Trp 188 and Trp 218 has been shown very rigid.
This work was supported by the Czech Science Foundation (Project 17-00726S).