デスレセプターを介するアポトーシス誘導に重要な 新規分子DELEの同定とその機能解析

Apoptosis is known as a form of programmed cell death, essential for the organogenesis of individuals, maintenance of the immune system, and elimination of injured cells and tumor cells. In cases when the regulatory mechanism of apoptosis becomes imbalanced, it induces severe diseases; for example, tumor development, breakdown of the immune system or neurodegeneration.Members of the death receptor (DR) family including tumor necrosis factor (TNF) receptor type1, Fas, DR3, TNF-related apoptosis inducing ligand receptors (TRAILR1/DR4 and TRAILR2/DR5) and DR6 are responsible to transduce apoptosis signal. These receptors are important for the regulation of many physiological and pathological events related to several human diseases. DR member family molecules induce apoptosis by the trimerization and aggregation of DR molecules on the plasma membrane. This receptor aggregation leads to recruitment of several subcellular proteins such as Fas-associated death domain protein (FADD) to the cytoplasmic domain of these receptors. The recruitment of FADD to DR is mediated through each death domain (DD) of DR to induce activation of caspases, cysteine proteases resulting in apoptosis induction.Death associated protein 3 (DAP3) is a GTP binding protein originally identified as a molecule which is responsible for activating interferon γ-induced apoptosis, and it has been reported that DAP3 is also important as a signal transducer for apoptosis induced by DR stimulation. In TRAIL induced apoptosis, DAP3 binds to TRAIL receptors, DR4 and DR5, and forms a complex with caspase-8 through FADD to activate caspase-8 depending on the stimulation of TRAIL. Thus, DAP3 is functional in cytoplasm for activation of TRAIL-mediated signaling pathway immediately downstream of DR4 and DR5. On the other hand, DAP3 is also observed abundantly in mitochondria, and the deletion mutant of DAP3 which lacks N-terminal region containing mitochondrial localization signal is not able to activate an apoptosis induced by the stimulation of FasL and TNF-α. These observations suggest that the mitochondrial localized DAP3 is also responsible for induction of apoptosis. However, molecular mechanisms of mitochondrial DAP3 for induction of apoptosis are poorly understood.From this point of view, initially, the mitochondrial localized proteins were selected by using the prediction program of subcellular localization (PSORT II; http://psort.ims.u-tokyo.ac.jp/) from positive clones obtained by yeast two-hybrid screening, previously performed in our laboratory to identify DAP3-binding proteins. The results showed that the subcellular localization of one of the positive clones which encoded a functionally unknown protein was predicted to be localized in mitochondria. Consequently, focused on this clone designated as DELE (death ligand signal enhancer), the molecular functions of DELE were analyzed. The DELE protein is a 55 kDa protein consists of 515 amino acids, and found to contain a mitochondrial targeting sequence at the N-terminus, two tetratrico peptide repeat (TPR) motifs. In this study, the molecular functions of DELE on the apoptosis induced by the stimulation of TNF-α, FasL and TRAIL were characterized. The co-immunoprecipitation analysis revealed that DELE actually binds to DAP3 in mammalian cells. The subcellular localization of DELE was analyzed by confocal laser scanning microscopy. The result shows that the subcellular localization of DELE is mainly observed in mitochondria, as was predicted by the analysis using PSORT II. The A549 cell lines in which the DELE gene is stably expressed were found to become susceptible to the apoptosis induction by the stimulation of TNF-α, anti-Fas, and TRAIL. In addition, knockdown of DELE gene expression by siRNA treatment significantly protected the HeLa cells from the apoptosis induction by the stimulation of these cytokines. Moreover, activation of caspase-3, -8, and caspase-9 by these stimulations was significantly suppressed by the knockdown of DELE gene expression.In conclusion, this study demonstrated that the newly identified DELE regulates the death receptor-mediated apoptosis through the regulation of caspase activity.