箆蔴子毒蛋白質促使計劃性細胞凋亡的分子機制之研究―人類B型血球抗原係一與箆蔴子毒蛋白質-A鏈結合的細胞凋亡調控者

Abstract

ABSTRACT Ricin is a member of type II ribosome-inactivating protein (RIP) family. It is composed of a toxophoric A-chain (RTA) and a galactose-binding B-chain (RTB). In cells, the RTA is retrograde translocated to one of its target site, endoplasmic reticulum (ER). Then, the RTA uses its RNA N-glycosidase activity to specifically remove an adenine residue (A4324) from the 28S rRNA, and inhibits protein biosynthesis. Several lines of evidence have shown that ricin induces cell death through two pathways: Inhibition of protein translation and inducing of apoptosis. A yeast two-hybrid screening using RTA catalytic mutant (E177Q, R180L) as bait is employed to identify RTA-binding proteins. Three clones are isolated during screening. Specific interactions between RTA and these clones are confirmed by yeast co-transformation and reporter assay. Nucleotide sequencing and data base analysis show that these clones encode partial Nrf3, hPLIC-2 and BAT3. Interestingly, they belong to the Ubiquitin-like protein family. The BAT3 is chosen as a major target in this study since 70 % of positive two-hybrid clones encode the same fragment of BAT3 (BAT3614-1044). The cDNA encoding BAT3614-1044 is sub-cloned in a mammalian expression vector and expressed in cells as N-terminal FLAG-tagged BAT3614-1044. The BAT3614-1044 displays both nuclear and cytoplasmic localization as revealed by immunocytochemistry and confocal microscopy. Ricin treatment causes cleavage of BAT3614-1044 and a cleaved fragment is generated. The cleavage is blocked by zVAD-fmk or zDEVD-fmk but not by calpain or serine protease inhibitor. Site-directed mutagenesis reveals that BAT3614-1044 is cleaved at the consensus caspase-3 cleavage site (DEQD1001↓G). To further explore roles of BAT3 in ricin-triggered apoptosis, full-length BAT3 cDNA is cloned by EST mapping or direct PCR from Jurkat cDNA library. A BAG domain-truncated variant of BAT3 due to alternative splicing is also obtained. Ectopically expressed BAT3 is cleaved by caspase-3 during treatment with ricin. The cleavage of BAT3 is inhibited by zDEVD-fmk. Endogenous BAT3 is also cleaved by ricin-induced caspase-3 and a C-terminal fragment with 131 amino acid residues is released (CTF-131). In caspase-3 deficient MCF-7 cells, BAT3 remains intact during treatment with ricin. BAT3 protein is localized in the nucleus, while CTF-131 is localized in the cytoplasm. By amino acid sequences analysis and site-specific mutagenesis, a novel nuclear localization signal in BAT3 is identified. Strikingly, the CTF-131 expressing cells display hallmarks of apoptosis including cell rounding, shrinkage, chromatin condensation and externalization of phosphatidylserine. CTF-