2010-08-012024-05-13https://scholars.lib.ntu.edu.tw/handle/123456789/642685摘要：之前我們實驗室發現了 一個新的基因’命名為NRIP (Nuclear Receptor Interaction Protein)，其為一雄性激素核接受器的共同轉錄因子，在雄性激素的刺激下，會增加雄性 激素受體提升PSA (Prostate Specific Antigen)的表現量(Tsai et al., 2005)，此外，我們也證 明了 NRIP本身藉由泛素/蛋白酶體增加雄性激素受體蛋白質的穩定性(Chen et al.,2008)。在此研究中，我們將廣泛的研究NRIP作為轉錄共同因子藉由去招集轉錄相關因 子及做為一特殊接合蛋白連接E2泛素接合酶來調控雄性激素受體蛋白的機制。近期的 報告指出，DDB2-DDB1-Cullin4-ROC1複合體含有對組織蛋白降解的E3接合酶功能來 活化核染色質DNA，有趣的是，在我們的初步研究發現DDB2為一新的可以與雄性激 素接受體蛋白相互作用蛋白(preliminary results, Figure 1)，並且可以降解雄性激素接受體 蛋白造成含有雄性激素接受體蛋白的細胞(LNCaP)生長速率的下降，但不會影響不含雄 性激素接受體蛋白的細胞(PC-3) (preliminary results Figure 2)，顯示DDB1可能參與雄性 激素接受體蛋白的降解作用，詳細的機制在此計畫會被更進一步的探討。在其他的研究者的報告中，NRIP作為Cul4-DDB1複合體的一員又被稱為DCAF6或 IQWD1，但是其功能尚未被確定。DDB1及NRIP均為含有WD40-repeat的蛋白，Cullin 4功能為載體而DDB1負責展現受質；Cul4-DDB1通常利用特殊的受質標定連接蛋白， 此類蛋白大部分含有WD40-repeat。我們的初步研究結果顯示利用NRIP純化前列腺癌 細胞(LNCaP)的核萃取物可以發現Cul4A、Cul4B及DDB1的存在(preliminary results, Figure 3A)，另外從 NRIP 與 DDB1 的相互作用(preliminary results, Figure 3B)的結果指出 NRIP可能參與Cul4-DDB1複合體，因此我們假設NRIP是藉由競爭 AR-DDB2-DDB1-Culin 4複合體來阻礙雄性激素受體蛋白的降解(Figure 4)。在此計畫中 我們也將釐清NRIP在雄性激素受體蛋白穩定性所扮演的角色及機制。由於NRIP可以增加雄性激素受體調控的轉錄活性，我們將檢查是否NRIP可以調控 核染色質重組，例如組織蛋白的修飾作用。由於NRIP含有可以與鈣離子/攜鈣素結合的 IQ結構域，而我們的初步結果也顯示NRIP可以與攜鈣素結合(Figure 3A)，失去IQ結 構域的NRIP則失去與攜i弓素的結合力(Preliminary results, Figure 5)，i弓離子/攜i弓素的下 游目標基因包括可以作用在核染色質重組的鈣調磷酸酶及攜鈣素激酶n，它們分別是磷 醋酶和激酶；舉例來說，攜i弓素激酶n被報導出可以稱酸化HDAC4而導致稱酸化的 HDAC4自核内輸出至細胞質中使得抑制數個基因的表現；除此之外，Cul4-DDB1複合 體也已被報導其功能不只做為E3接合酶還可參與組織蛋白的曱基化進而調控基因的表 現，因此我們想探討NRIP做為一轉錄共同因子在調控核色質重組上的角色。NRIP在生物活體上的研究，我們已建構了 NRIP的基因剔除小鼠(preliminary results, Figure 6)做為我們研究的模式生物，NRIP是一個可被雄性激素所調控的基因， 而前列腺是一個典型依靠雄性激素的器官，我們初步研究顯示在NRIP基因剔除鼠(10 週)前攝護腺葉(AP)有未分化及細胞增生現象(Figure 7A及B)。如同Nkx3-1可以調控前 列腺表皮組織分化，且是一個已被清楚了解可被雄性激素調節其表現的基因，其在前列 腺的發育以及癌症上扮演重要的角色。因此，我們將會利用NRIP的基因剔除鼠做為模 式研究NRIP在前列腺發育及癌症發生上所扮演的角色。研究目標：1.NRIP在雄性激素受體蛋白穩定上所扮演的角色：我們將釐清雄性激素受體蛋白的降 解藉由DDB2-DDB1-Cul4-ROC1的模式，之後再確定NRIP在此複合體中是藉由阻礙 DDB1或DDB2來達到穩定雄性激素受體蛋白2.NRIP在核染色質重組的機制上來增強雄性激素受體所調控的基因轉錄：NRIP含有可與攜鈣素結合的IQ結構域，我們有興趣的是NRIP是否可經由與攜鈣素的結合本身、 或活化鈣調磷酸酶或攜鈣素激酶n來調控組蛋白修飾導致的核染色質的重組（例如 曱基化或乙酰化）3. NRIP在前列腺分化及攝護腺癌形成上所扮演的角色：利用NRIP的基因剔除鼠做為 模式生物廣泛地研究NRIP在前列腺的發育上以及攝護腺癌的形成上所扮演的角色<br> Abstract: Previously, we found a novel gene, nuclear receptor interaction protein (NRIP) thatcan directly interact with AR and functions as a transcription cofactor to enhance anAR-driven prostate specific antigen (PSA) gene expression (Tsai et al., 2005). Plus we alsoidentified that NRIP is a novel AR-target gene and protects AR protein stability fromubiquitine/proteosome degradation (Chen et al., 2008). The major goal of this project is toinvestigate how NRIP serving as transcription cofactor to recruit transcription-related factorsand as a specific adaptor for preventing the recruitment of E3 ligase complex to appropriatetargets (such as AR), respectively. Recent reports demonstrated thatDDB2-DDB1-Cullin4-ROC1 complex contains E3 ligase function for histone degradation toactivate chromatin DNA. Intriguingly, our preliminary results found that DDB2 is a novelAR-interaction protein (preliminary results, Figure 1) and can degrade AR resulting in thedecreased growth rate in AR-containing cells (LNCaP) but not in AR-null cells (PC-3)(preliminary results Figure 2), implying that DDB2 may involve in AR protein degradation.The detailed mechanisms of DDB2-degrading AR will be further investigated in this project.NRIP is also named DCAF6 or IQWD by the other researchers who reportedDCAF6/IQWD bounded with Cul4-DDB1 complex, but not defined its function. Both DDB1and NRIP are WD40-repeat proteins. Culin 4 functions as dock and DDB1 as substratepresenting function; Cul4-DDB1 usually uses specific substrate-targeting adaptors most ofwhich are WD40-repeating protein. Our preliminary results show that Cul4A, Cul4B, andDDB1 are the proteins by NRIP pull-down with the nuclear extracts of HeLa cells(preliminary results, Figure 3A), plus the interaction between NRIP and DDB1 (preliminaryresults, Figure 3B), indicating that NRIP may participated DDB1-Cul4 complexes. Wethereby hypothesize that NRIP competes AR-DDB2-DDB1-Culin 4 complexes to preventfrom AR degradation (Figure 4). The detailed mechanisms of NRIP role for AR proteinstabilization will be pursuit in this project.As to the role of NRIP in enhancing AR-mediated transcriptional activity, we willexamine whether NRIP can regulate chromatin remodeling such histone modification. SinceNRIP contains IQ domain which is calcium/calmodulin binding domain. Our preliminaryresults show that NRIP could bind calmodulin (Figure 3A), deletion of IQ domain in NRIPlost the binding affinity of calmodulin (Figure 5). Calmodulin itself or its downstreamtargeted genes such as calcineurin and calmodurin kinase II are reported to involve inchromatin remodeling. Calcineurin and calmodurin kinase II are identified as phosphatase andkinase to function for chromatin remodeling, respectively. For example, calmodurin kinase IIis reported to phosphorylate HDAC4, and the the phosphorylated-HDAC4 exportes from thenucleus into cytosol which resulting in the depression of several genes’ transcription. Inaddition, the core CUL4–DDB1 complex has been reported to function not only E3 ligasefunction but also involved in histone methylation to regulate gene transcription. It isinteresting to examine the role of NRIP as a transcriptional cofactor to regulate chromatinremodeling.For further investigating the function of NRIP in vivo, we generated NRIP knock out(KO) mice as a model for study (preliminary results, Figure 6). NRIP is anandrogen-regulated gene, and the prostate is a classically androgen dependent organ Ourpreliminary results show that abnormal proliferation and/or differentiation of epithelial cellsin the anterior prostate in NRIP KO mice at 10 weeks in comparison with wt mice (Figure 7Aand 7B). Similarly, the Nkx3-1 gene is well-studied transcriptionally regulated by androgensand regulates prostate epithelial differentiation, and Nkx3.1 plays an important role in prostatedevelopment and cancer [Bhatia-Gaur et al.,1999; Wang et al.,2009]. Therefore we willinvestigate whether NRIP involving in prostate development and carcinogenesis. Knock-outNRIP mouse model to the role of NRIP in prostate development and prostate cancer formationwill be investigated. Therefore, three major approaches involved in NRIP role for AR proteinprotection, NRIP role for transcription regulation, and NRIP role in prostate development andcarcinogenesis will be accessed to further detail the mechanisms of NRIP function.Specific aims:1. NRIP role for AR protein protection. The model of DDB2-DDB1-Cul4-ROC1 for ARprotein degradation will be investigated. After that, NRIP role for interrupting in DDB1 orDDB2 of these complexes to protect AR protein will be identified.2. NRIP role for chromatin remodeling to enhance AR-driven gene expression.NRIP contains IQ domain which is responsible for calcium/calmodulin binding. It isinteresting to examine the NRIP interacting with calmodulin or activating calcineurin andcalmodurin kinase II to regulate chromatin remodeling such as histone modification(methylation and acetylation etc).3. NRIP role for prostate differentiation and prostate cancer formation. Using knock-outNRIP mouse model to study the role of NRIP in prostate development and prostate cancerformation will be comprehensively investigated.NRIP/DCAF6/IQWD1雄性激素受體攜鈣素Culin 4-DDB1複合體攝護腺發育NRIP/DCAF6/IQWDARcalmodulinCulin 4-DDB1 complexprostate development