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

Studies on the Molecular Mechanism of Ricin-triggered Programmed Cell Death—BAT3, a Ricin-A Chain Binding Apoptotic Regulator

吳渝樺(Yu-Hauh Wu)

指導教授 ： 林榮耀

國立臺灣大學/醫學院/生物化學暨分子生物學研究所/博士(2004年)

https://doi.org/10.6342/NTU.2004.01024

摘要

摘要
箆蔴子毒蛋白質屬於第二型核醣體去活性蛋白質家族的一員。箆蔴子毒蛋白質係由一具酵素活性的A-鏈 (RTA) 及一能與半乳醣結合的B-鏈 (RTB) 所組成。RTA與RTB之間以一對雙硫鍵互相連結。RTB與細胞表面含半乳糖單元的接受器結合後將導致整個箆蔴子毒蛋白質分子藉由內噬作用進入細胞。在細胞中，RTA被逆向轉運至內質網。之後，RTA利用其RNA N-醣苷水解酶活性專一性地將28S核醣體RNA上第4324號腺口票口 令 移除，並抑制蛋白質的生合成。
近年來釵h研究結果顯示箆蔴子毒蛋白質經由以下兩種途徑造成細胞死亡：抑制蛋白質轉譯反應及誘發計畫性細胞凋亡。為了要探索與箆蔴子毒蛋白質造成計畫性細胞死亡的相關分子，酵母菌雙雜合篩選系統在本研究中被用於找尋細胞內的RTA結合蛋白質。經過完整的篩選過程，我找到了三種RTA結合蛋白質。經由核苷酸序列分析及資料庫比對證實此三種純株分別可編譯出Nrf-3, hPLIC-2 及 BAT3 的一個片段 (BAT3614-1044)。有趣的是這三種RTA結合蛋白質皆屬於類ubiquitin 蛋白質家族的成員。因為由酵母菌雙雜合篩選系統中得到的RTA結合蛋白質中，以BAT3614-1044佔70 ﹪最多，所以本研究著重於探討BAT3在箆蔴子毒蛋白質造成計畫性細胞死亡中所扮演的關鍵角色。為了研究BAT3614-1044 在細胞中的位置及弁遄ABAT3614-1044 的cDNA被選殖入真核細胞表現載體中以利產生具有氮基端FLAG標籤的BAT3614-1044。經由免疫細胞染色及共軛焦顯微鏡觀察，發現BAT3614-1044均勻地分佈於細胞核及細胞質中。將細胞以處理箆蔴子毒蛋白質處理後，可造成BAT3614-1044被水解出一個片段。廣效型細胞凋亡酶(caspase)抑制劑及專一性第三型細胞凋亡致效器(caspase-3)抑制劑均可有效阻斷BAT3614-1044被水解，然而，依鈣蛋白質分解酵素或絲胺酸蛋白酶抑制劑無法阻斷BAT3614-1044被水解。 利用定位突變法成它a證實BAT3614-1044被切在符合第三型細胞凋亡酶的辨識序列(DEQD1001↓G)上。
為了進一步探討BAT3在箆蔴子毒蛋白質造成細胞死亡中所扮演的角色，全長的BAT3 cDNA經由EST mapping及直接以基因庫為模板的PCR反應所選殖。此外，一個BAG弁鈰炾麈吤◥慷AT3變異株也成它a被選殖。在箆蔴子毒蛋白質處理下，BAT3被第三型細胞凋亡酶所切割。以zDEVD-fmk前處理可抑制第三型細胞凋亡酶將BAT3水解的反應。
內生型的BAT3亦會被箆蔴子毒蛋白質所誘發的第三型細胞凋亡酶所切割，且產生一個羰基端131個胺基酸的片段(CTF-131)。在第三型細胞凋亡酶缺失的人類乳癌細胞株(MCF-7)中，BAT3在箆蔴子毒蛋白質處理下維持完整不被水解。完整的BAT3位於細胞核中；然而CTF-131則位於細胞質中。藉由胺基酸序列分析及定位突變法，一個新的核定位序列(Nuclear localization sequence)被鑑定出來。令人印象深刻的是，CTF-131表現後造成計畫性細胞凋亡的特徵包括細胞質皺縮圓起、染色質緊緻化及磷酯絲胺酸外露至細胞膜外。由CTF-

關鍵字

細胞凋亡 ； 箆子毒蛋白質-A鏈 ； 人類B型血球抗原 ； 蔴

並列摘要

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-

並列關鍵字

Programmed Cell Death ； BAT3 ； apoptosis ； Ricin-A Chain

參考文獻

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