第壹部份：人類B型血球抗原BAT3片段之氮端53個殘基引發細胞"hummingbird"型態變化之功能性研究；第貳部份：人類肝癌致病基因Granulin之功能性研究

Part Ⅰ: Functional studies of a N-terminal 53-residues of BAT3 CTF-131 fragment inducing hummingbird morphological change Part Ⅱ: Studies on mutated Granulin, identified from a full-length cDNA library of hepatocellular carcinoma

李宏仁(Hong-Jen Lee)

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/碩士(2004年)

https://doi.org/10.6342/NTU.2004.01020

摘要

第壹部分論文摘要

蓖麻子毒蛋白 (ricin)，是屬於第二型核糖體去活性蛋白質，在1880年由Stillmark自大戟科蓖麻屬植物 (Ricinus communis) 的種子萃取而得。蓖麻子毒蛋白是由一條A鏈 (RTA) 及一條B鏈 (RTB) 所組成，並且藉由B鏈的親醣蛋白質活性 (lectin)，與細胞表面上的受體結合，使得蓖麻子毒蛋白得以順利透過內噬作用 (endocytosis) 進入細胞，執行其毒殺作用。RTA具有RNA N-糖苷鍵水解酵素 (N-glycosidase)的活性，在進入細胞後，可以去除28S 核糖體RNA上第4,324位置的adenine residue，使得核糖體失去正常活性，進而抑制蛋白質的生合成，達到毒殺細胞的作用。
為了研究蓖麻子毒蛋白毒殺細胞的作用機制，本實驗室之前利用RTA作釣餌，以酵母菌雙雜合系統 (yeast two-hybrid) 的實驗方法，找到一個名為人類第三型白血球抗原B關聯性轉錄物 (human HLA-B-associated transcript 3)，又簡稱為BAT3。利用遺傳工程，分子生物學技術及細胞生物學的研究方法，我們發現對細胞加入蓖麻子毒蛋白之後，會使細胞產生細胞凋亡 (apoptosis) 的表徵。除此之外，BAT3會被蓖麻子毒蛋白所活化的caspase-3切割，產生一段由C端131個胺基酸所組成的片段 (CTF-131)。
在研究CTF-131引發細胞凋亡的過程中，意外的發現，如果在哺乳動物細胞中，只表現CTF-131的氮端53個胺基酸 (N53)，所引發的細胞表徵，和CTF-131迥然不同。研究結果顯示，在哺乳動物細胞中大量表現N53時，細胞型態會被拉長，使得細胞變得類似蜂鳥狀 (hummingbird phenotype)。
在本研究中，我們利用分離細胞中F-/G-actin的方法，証實了在N53表現的細胞中，F-actin確實有大量累積的現象。此一結果，和之前利用共軛焦顯微鏡所觀察到的現象，是一致的。為了更進一步研究N53引發細胞型態改變的機制，我們以N53為釣餌，利用酵母菌雙雜合系統 (yeast two-hybrid) 的實驗方法，試圖找出和N53有交互作用的蛋白質。在此實驗策略之下，我們找到了一個名為coronin的蛋白質。根據之前發表的研究報告指出，coronin會和肌動蛋白 (actin) 產生交互作用，並且在酵母菌中，coronin會促進F-actin 的聚合作用 (polymerization)。而N53和coronin在引發細胞變型的機制中，扮演何種角色，則需更進一步的實驗去加以探討。

第貳部分：論文摘要

過去的研究顯示，人類十大死因中，癌症往往是名列前矛。在各式各樣的癌症中，肝癌對人類的影響，又更為嚴重。和已開發國家相比較，肝癌在亞洲地區的發生率更高。在台灣，肝癌的致死率在各種癌症中，排名第一。目前已知，肝癌的發生，牽涉到許多的因子，例如: 環境中的致癌物，酒精，病毒的感染，或是遺傳因子造成。而且，這些影響因子，彼此之間的關係又相當複雜。
近年來，隨著生物技術的開發，各種大規模的篩選致癌基因的研究，如雨後春筍般的展開。例如:有科學家們利用DNA microarray 或是comparative genomic hybridization要試圖找出，促使肝癌發生的特殊基因。在本實驗室中之前的研究中，自一個由C型病毒 (hepatitis C virus)感染的肝癌組織中，建構了一個全長互補DNA資料庫 (full-length cDNA library)。並且，將這資料庫中所有的DNA序列，經由定序確認後，和NCBI的資料庫比對，找到了24個有缺陷的基因，其中，包括了granulin。
Progranulin由593胺基酸組成，包含了七個半的granulin repeats。由於醣化修飾 (glycosylation)的作用，使得其分子量在經由西方墨點轉漬法鑑定時，有接近9萬道耳吞 (90 kDa)。我們在肝癌組織中，找到的是有缺失的progranulin (deletion genotype)。為了研究Progranulin在肝癌中所扮演的角色，我們試圖用colony formation assay來觀察。藉由這個方法，我們發現，野生型的progranulin (wild type progranulin)比突變型的progranulin更具有使動物細胞轉型 (transformation)的能力。至於progranulin的表現量和肝癌發生之間的關係，則有待進一步的研究証實。

關鍵字

肝癌 ； 細胞型態 ； B型血球抗原 ； 全長cDNA資料庫

並列摘要

PartI：Abstract

Ricin, consists of A chain (RTA) and B chain (RTB), is a toxic protein which was isolated from the seeds of the castor beans Ricinus communis. It is a member of type Ⅱ ribosome-inactivating protein (RIP). The RTA possesses a RNA N-glycosidase activity which can inactivate ribosomes by removing the 4324th adenine residue from the 28S rRNA and inhibit protein synthesis to cause cell death. Apoptosis, also named program cell death, was found in ricin-treated HeLa cells.
By using RTA as bait in a yeast two-hybrid system, a RTA-interacting protein was isolated and designed as human HLA-B-associated transcript 3 (BAT3) in the previous study. It had been demonstrated that BAT3 was cleaved by ricin-induced caspase-3 activity and released a c-terminal 131-residue fragment (CTF-131). Overexpression of the CTF-131 fragment in HeLa cell causes several characteristic apoptotic morphologies such as phosphatidylserine (PS) exposure, cell rounding and shrinkage, nuclear condensation and actin disruption. It was identified that CTF-131 contains a Bcl-2-associated athanogene (BAG) domain and this BAG domain was responsible for inducing the apoptotic morphologies. Interestingly, we identified a fragment, the N-terminal 53 amino acids (N53) of CTF-131 (1002-1054 residues), which had dramatic effects on the cell morphology and led to distinct morphologic changes of cells which resembled the “hummingbird” phenotype.
By fractionation of F-/G-actin using sucrose density gradient centrifugation, F-actin accumulation was discovered in the N53 transfectants but not in those control groups. The results suggest that the elongated cell type resulted from N53 transfection could be due to the accumulation of F-actin.
To study the roles of the N53 in the morphologic changes and accumulation of F-actin, identification the proteins interacting with N53 was carried out by using yeast two-hybrid system, and to study whether these interacting proteins involved in morphologic change. An actin binding proteins, coronin, was found to be the N53 interacting protein. It suggests that N53 interacted with coronin and caused the decrease of dissociation of F-actin.

PartII：Abstract

Hepatocellular carcinoma (HCC) is one of the most common human malignant tumors in the world. The disease is highly prevalent in Asia but relatively rare in developed countries. In Taiwan, it ranks first in terms of cancer mortality. Different aetiological factors such as hepatitis viral infection , dietary aflatoxins, or chemical carcinogens are associated with the development of liver cancer. Nevertheless, the molecular mechanism remains to be clarified.
A full-length cDNA library of HCC was constructed from a 65-year-old male patient infected by hepatits C virus. The sequencing data were collected and analyzed for their sequence similarity against the GeneBank nucleotide library maintained by the NCBI. By the BLAST program, we had got 24 deletion clones, including Granulin (GRN). GRN was identified as a putative growth factor which is characterized by a unique and highly conserved cysteine-rich motif of 12 cysteines. Cloning of the cDNA for the GRN showed that it encoded a 63 kDa protein that contained a signal peptide (designed as progranulin, proGRN). Previous investigation indicates that expression of GRN in cancer cells may play a critical role in tumor formation.
In this study, the mutated GRN is deleted in-frame from 331-465 base pair in its coding region (shortened 135 bps). This mutated GRN was cloned, and wild type GRN was cloned into pcDNA3 vector for mammalian cells transfection. In colony formation assay, wild type proGRN conferred SW 13 cells better ability of anchorage-independent growth. Further study will be needed to elucidate the relationship between the proGRN and HCC.

並列關鍵字

coronin ； hummingbird phenotype ； BAT3 ； hepatocellul

參考文獻

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