- 學位論文
NRIP穩定雄性激素受體蛋白以及增進雄性激素受體轉錄活性之機制探討
Investigation of the mechanism of NRIP-stabilizing AR protein and enhanced AR transcription activity
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摘要
核受體交互作用蛋白NRIP (nuclear receptor interaction protein)為本實驗室利用雄性激素受體(Androgen receptor, AR)的C端作餌，以yeast-two hybrid 選殖系統所鑑定而得；實驗室先前的研究發現：NRIP不但可以直接與AR交互作用，並且也是一個AR共同活化子，在雄性激素存在之下能增強AR下游的Prostate-specific antigen(PSA)轉錄活性。在前列腺細胞株LNCaP抑制NRIP表現，會使得細胞進行細胞凋亡，這個現象顯示NRIP在前列腺癌細胞存活上扮演重要的角色。 最近，本實驗室發現NRIP是一個AR所調控的基因，不但在有雄性激素存下可以增加NRIP的轉錄，且NRIP的promotor 具有雄性激素反應區域（androgen response element）。此外，本實驗室也發現NRIP可以穩定AR蛋白。在NRIP表現被抑制時會導致AR經由26Ｓ proteasome降解。於是，本論文主要目標是要更進一步探討NRIP穩定AR蛋白的機制。 據現階段的研究知道DDB1-Cullin4複合體具有E3接合酶的作用，能夠促使各種蛋白的降解，而DDB1和NRIP兩者皆具有WD40 domain。由DDB1的pull down的實驗中發現NRIP會和DDB1交互作用；因此，DDB1-Cullin4路徑是否牽涉NRIP穩定AR蛋白的機制值得深入探討。本實驗首先利用co-IP實驗證實NRIP和DDB1的交互作用；利用site-directed mutation製備NRIP突變蛋白後，標定出上負責和DDB1作用的motif。此外，也發現一個參與DNA損傷修補作用的DDB2蛋白可與AR進行交互作用，並在細胞中表現DDB2時，AR蛋白隨即減少。而在探討NRIP如何穩定AR蛋白的機制,我們利用in vivo competition assay 證明NRIP有可能透過與DDB2競爭對AR的結合，使得AR蛋白被穩定下來。另外，我們利用失去和DDB1結合能力的突變型NRIP送到LNCaP細胞中，發現突變型的NRIP仍保有增加AR下游基因調控的轉錄活性，證實NRIP調控AR轉錄活性不是透過DDB1-CUL4這條路徑。 而最後，為了在動物模式中研究NRIP的功能，我們建構了NRIP基因剔除小鼠，並且在DNA, RNA 以及蛋白質的層次上皆證明小鼠已缺失NRIP。此外，我們分離出NRIP基因剔除胚胎纖維母細胞，表現NRIP可以增加AR蛋白的表現量，並且我們發現在MEF細胞中發現NRIP與細胞生長有密切相關。未來，我們將觀察NRIP基因剔除所引起的表徵，並更進一步研究NRIP在生物體中扮演的角色。
並列摘要
By using yeast-two hybrid system, Nuclear Receptor Interaction Protein (NRIP) is found in our lab. NRIP is an AR coactivator which can directly interact with AR. It can activate prostate-specific antigen (PSA) transcription in an androgen-dependent manner. When knockdown of NRIP in LNCaP cell causes cell apoptosis that reveals a vital role of NRIP in cell survival in prostate cancer cell. Recently, we found that NRIP is also a novel AR target gene. Transcription of NRIP can be increased in the presence of androgen, and then we identified the androgen response element in the promoter of NRIP. Furthermore, we found the role of NRIP in AR protein stability .Where the knockdown of NRIP contributes to AR degradation through proteasome. DDB1-Cullin4 complex contains E3 ligase function for various proteins’ degradation. From DDB1 pull down assay, it was reported that NRIP is one of DDB1 interaction proteins, therefore we are interested to examine the fact whether NRIP can bind to DDB1. The interaction between NRIP and DDB1 was confirmed by reciprocal co-immunoprecipitation .We found that ＷDxR motif on NRIP responsible for DDB1 binding was identified using site-direct point mutation. When knockdown DDB1 or CUL4A, we observed an enhancement of AR protein level in LNCaP cells. When ectopic expression of NRIP can reduce AR protein ubiquitin level. Hence, we hypothesize NRIP-stabilizing AR protein involved in DDB1-Cul4A complex. DDB2 is a well-known WD40 protein that associates with DDB1. DDB1-DDB2 heterodimer was involved in NER pathway which UV-induced DNA damage repairs. We found that DDB2 can associated with AR and introduced DDB2 can reduce AR protein level in LNCaP cell. This degradation of AR protein is dependent on presence of DHT. To further demonstrate the mechanism of NRIP-stabilizing AR protein; we used in vivo competition assay to indicate the mechanism of NRIP-stabilizing AR. The mechanism of NRIP-stabilizing AR may be through competition with DDB2 for binding to AR. Furthermore, we introduced mutant NRIP (can not interact with DDB1) into LNCaP cell to observe the PSA gene expression, the result excluded the DDB1-CUL4 pathway in the mechanism of NRIP by which contributes to enhance AR mediated transcription activity. To approach investigating the function of NRIP in vivo, we had generated NRIP knockout mice and confirmed the genotype of NRIP KO mice in DNA, RNA and protein level. Based on the role of NRIP in AR stability in cell culture, we observed an enhancement of AR protein when introducing NRIP in NRIP KO MEF cell. Moreover, the proliferation of KO MEF cell was slower than wild-type cell. It suggests that NRIP may play a role in cell growth. In the future, we would investigate more functions of NRIP in vivo using knockout mice model.
參考文獻
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延伸閱讀
- Hsu, C. Y. (2006). 蛋白酶激活接受器ㄧ與下游G蛋白α次單元及細胞外信號調節激酶在富含窖蛋白的分層中位置與分布之研究 [master's thesis, National Tsing Hua University]. Airiti Library. https://www.airitilibrary.com/Article/Detail?DocID=U0016-1303200709321165
- Hsieh, W. Y. (2005). c-Maf 及其交互作用蛋白調節細胞激素表現之探討 [master's thesis, National Taiwan University]. Airiti Library. https://doi.org/10.6342/NTU.2005.02206
- Wang, S. H. (2011). 性激素調控B型肝炎病毒mRNA轉錄過程之分子機制研究 [doctoral dissertation, National Taiwan University]. Airiti Library. https://doi.org/10.6342/NTU.2011.10185
- Chien, J. Y. (2011). Functional Characterization of RNA Polymerase II Subunit RPB4 in Transcription and mRNA Decay Machinery [master's thesis, National Taiwan University]. Airiti Library. https://doi.org/10.6342/NTU.2011.02074
- Chen, B. (2010). Structural and functional characterization of human APPL2, a novel adaptor protein involved in insulin signaling [master's thesis, The University of Hong Kong]. Airiti Library. https://www.airitilibrary.com/Article/Detail?DocID=U0029-1812201200018498