Identification And Analysis Of Subcellular Localization Signal Of Caspase-3

Apoptosis is a spontaneous process of programmed cell death in the physiological or pathological condition, also a normal event in body development and maintenance of tissue homeostasis,and occurs under the tight control of the body. The apoptotic pathway is classified as extrinsic and intrinsic pathways. The extrinsic pathway is initiated by ligation of a trans-membrane death receptor with their ligands, for instance, ligation between Fas and FasL;TNF and TNFR1. Upon ligation, the Fas or TNFR1 forms microaggregates at the cell surface, then recruiting the adaptor protein FADDs by virtue of interaction with their death domains(DDs),constructing the FasL-Fas-FADD or TNF-TNFR1-FADD。Both are named as death-inducing signal complexes (DISC). It is within this DISC that the initiator caspase-8 is recuited by homophilic interaction between their death effector domains(DEDs), and activated. Then the activation of executioner caspase-3 is carried out by activated caspase-8, leading to cell apoptosis. The intrinsic pathway is used to climinate cells in response to ionizing radiation, chemotherapeutic drugs and mitochondrial damage. Following the death trigger, cytochrome C is released from mitochondria by proapoptotic factors of Bcl-2 family, and binds to Apaf1 and ATP/dATP. cytC-Apaf1-ATP/dATP complex is named'apoptosome', whose central component is Apaf1 which recruits caspase-9 via its caspase recruitment domain(CARD). Then the recuited caspase-9s activates thenselves, this is followed by the same executioner caspase activation as the extrinsic pathway.Caspase-3 is shared by the two pathways and a key regulating protein in cell apoptosis. During apoptosis, caspase-3 is cleaved and activated by the proteases, and enters into the nucleus, resulting in apoptotic biochemical and morphological changes. However, the molecular mechanism of nuclear-entering of active caspase-3 is still unknown. Interchange of materials between the cytoplasm and the nucleus occur through dedicated transport channels crossing the nuclear envelope, the nuclear pore complexes(NPCs). Macromolecules such as protein transporting through NPC need nuclear localization signal(NLS) or nuclear export signal(NES). Identification of subcellular localization signal in the caspase-3 molecule is important to understand the process.In this study, we constructed various truncated and fused with GFP caspase-3 mutants, observed the cellular location of these mutants, and determined the subcellular localizations signal in these mutant caspase-3 molecules. The results show that caspase-3 lacks obvious nuclear localization signal (NLS), but bears an obvious nuclear export signal (NES), which is located at the C-terminus of the caspase-3 small subunit including residues 220~245.