Please use this identifier to cite or link to this item:
http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/43287
Title: | NESI蛋白質與其核膜上結合蛋白質之功能分析 Functional analysis of NESI and its interacting proteins on nuclear envelope |
Authors: | Jia-Yin Jiang 姜佳吟 |
Advisor: | 張明富(Ming-Fu Chang) |
Keyword: | D型肝炎病毒,大型delta抗原,NESI蛋白質,核輸出,核孔複合體, hepatitis delta virus,large delta antigen,nuclear export signal-interacting protein,nuclear export,nuclear pore complex, |
Publication Year : | 2009 |
Degree: | 碩士 |
Abstract: | D型肝炎病毒(hepatitis delta virus, HDV) 為B型肝炎病毒的衛星病毒,必須獲得B型肝炎病毒的外套膜才具有感染力。藉由與B型肝炎病毒的共同感染(co-infection)或重疊感染(super-infection)造成肝臟病變。D型肝炎病毒外觀為球狀具有外套膜且包含負向單股環狀RNA基因體,其全長約為1.7 kb。目前所知D型肝炎病毒的delta抗原是唯一由D型肝炎病毒所轉譯產生的蛋白質,可依大小分為兩型:分別是與病毒複製有關的小型delta抗原(small delta antigen, HDAg-S;195個胺基酸,約24 kDa);以及與病毒顆粒的包裹有關的大型delta抗原(large delta antigen, HDAg-L;214個胺基酸,約27 kDa)。
先前本實驗室的研究發現,大型delta抗原C端具有一段proline-rich的核輸出訊號(nuclear export signal;NES),將其命名為NES(HDAg-L)。此核輸出訊號是透過CRM1-independent pathway來調控大型delta抗原的核輸出。此外,宿主細胞中之NESI蛋白質(nuclear export signal-interacting protein)可與NES(HDAg-L)結合,參與大型delta抗原的核輸出與D型肝炎病毒的顆粒包裹。NESI蛋白質具有467個胺基酸(約53 kDa),經由序列的分析得知,其第4到第13個胺基酸可能與肌動蛋白(actin)結合,第193到第209個胺基酸則具有bipartite nuclear localization signal。但是NESI蛋白質在宿主細胞內所扮演的角色,與如何影響大型delta抗原的核輸出的機制尚不清楚。 本研究的目的在深入了解NESI於細胞內蛋白質進出核膜的過程中所扮演的角色以及功能。首先利用免疫沉澱以及質譜儀分析方法,發現在細胞中lamin A/C可能與NESI蛋白質有交互作用。更進一步利用免疫沉澱與西方墨點法分析,證實兩者間專一性的交互作用。另外,以免疫螢光染色方法在共軛焦顯微鏡觀察下,發現NESI蛋白質和lamin A/C在細胞核膜上有共位的現象。而GST pull-down的實驗分析亦顯示NESI蛋白質的N端31至116個胺基酸的區域即具有與lamin A/C結合的能力;在蔗糖梯度離心的實驗中也顯示,NESI、lamin A/C以及帶有phenylalanine- glycine repeats的nucleoporins可存在於同一個複合體中。 為了更進一步探討NESI蛋白質與lamin A/C的交互作用是否會影響大型delta抗原的核輸出,本研究進一步以shRNA降低NESI與lamin A/C蛋白質的表現量,再觀察D型與B型類病毒顆粒組裝及分泌情形。實驗的結果顯示,當NESI或lamin A/C的蛋白質表現量降低時,大型delta抗原的核輸出及D型肝炎病毒的組合會受到抑制,但對B型肝炎病毒表面抗原之類病毒組合則沒有影響。綜合以上結果,可以推論在核輸出及組合的過程中,NESI、lamin A/C以及帶有phenylalanine- glycine repeats的nucleoporins可能在核膜上形成一個複合體。但是此複合體在大型delta抗原核輸出及HDV類病毒顆粒的包裹的過程中扮演何種角色,需要更進一步釐清。 Hepatitis delta virus (HDV) is a satellite virus of hepatitis B virus (HBV) that requires HBV envelope proteins for packaging. HDV causes liver damage by co-infection or superinfection with HBV. HDV is a spherical RNA virus that contains a single-stranded circular RNA genome of 1.7 kb in length. Hepatitis delta antigen (HDAg) is the only known protein of HDV. There are two forms of delta antigen, the small HDAg (HDAg-S, 195 a. a., 24 kDa) and the large HDAg (HDAg-L, 214 a. a., 27 kDa). The HDAg-S facilitates the replication of HDV RNA, whereas the HDAg-L is required for the viral assembly. Previous studies from our laboratory identified a novel proline-rich nuclear export signal at the C-terminus of HDAg-L, designated NES(HDAg-L). The NES(HDAg-L) mediates the nuclear export of HDAg-L via a chromosome region maintenance 1 -independent pathway. In addition, a cellular factor NES(HDAg-L)-interacting protein (NESI) was identified to be involved in the nuclear export of HDAg-L and the assembly of HDV. NESI consists of 467 amino acid residues. Sequence analysis of NESI revealed a putative actin-binding site from amino acid residues 4 to 13 and a bipartite nuclear localization signal from amino acid residues 193 to 209. The role of NESI involved in nuclear export is not fully understood. In this study, mechanisms involved in the NESI-mediated nuclear export and possible interactions between NESI and nuclear envelope proteins were investigated. By performing immunoprecipitation followed by LC-MS/MS analysis, lamin A/C was identified to interact with NESI. The specific interaction between NESI and lamin A/C was further confirmed by Western blot analysis following immunoprecipitation. In addition, colocalization of NESI and lamin A/C on the nuclear envelope was demonstrated by confocal microscopy. GST pull-down assay demonstrated that the N-terminal 31 to 116 amino acid residues are required for NESI to interact with lamin A/C. Sucrose gradient centrifugation analysis implicated that NESI, lamin A/C and the phenylalanine-glycine nucleoporins may be present in the same complex on nuclear envelope. To investigate the functional role of NESI involved in HDAg-L-associated nuclear export, shRNA to NESI or lamin A/C applied in an HDV assembly culture system. The results demonstrated that both shRNAs to NESI and lamin A/C specifically inhibited HDAg-L nuclear export and HDV assembly, but had no effect on the assembly of the small surface antigen of HBV. Taken together, these results indicate that NESI may form complexes with lamin A/C and the phenylalanine-glycine nucleoporins for nuclear export. Detailed mechanisms of the complex involved in the nuclear export on nuclear envelope need further studies. |
URI: | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/43287 |
Fulltext Rights: | 有償授權 |
Appears in Collections: | 生物化學暨分子生物學科研究所 |
Files in This Item:
File | Size | Format | |
---|---|---|---|
ntu-98-1.pdf Restricted Access | 2.81 MB | Adobe PDF |
Items in DSpace are protected by copyright, with all rights reserved, unless otherwise indicated.