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標題: | NRIP透過質固醇受體與E2病毒蛋白調控人類乳突瘤狀病毒的基因表現 NRIP Enhances HPV Gene Expression via Interaction with Either GR or E2 |
作者: | Szu-Wei Chang 張思為 |
指導教授: | 陳小梨(Show-Li Chen) |
關鍵字: | 核受質結合蛋白,人類乳突瘤狀病毒,質固醇受體,E2蛋白, NRIP,HPV,GR,E2, |
出版年 : | 2011 |
學位: | 博士 |
摘要: | 我們之前發現了一個基因命名為核受體交互作用蛋白(Nuclear receptor interaction protein, NRIP),在質固醇受體(Glucocorticoid receptor, GR)及人類乳突瘤狀病毒(Human papillomavirus, HPV)的E2蛋白所驅動的基因表現上,其功能可作為一轉錄作用輔因子。我們闡述了NRIP為一個新的可以與人類乳突瘤狀病毒16型之E2結合蛋白,人類乳突瘤狀病毒16型的E2蛋白可以在生物體內、體外與NRIP直接形成一複合體,NRIP藉由其N端與E2蛋白的轉錄活化模組(Transactivation domain)相互作用,只有完整全長的NRIP可以穩定E2蛋白並誘導人類乳突瘤狀病毒的基因表現。利用兩種針對NRIP設計的小干擾RNA可以降低E2的表現及其所驅動的基因表現;我們另外發現在鈣離子存在的環境下,NRIP可透過它的IQ模組直接與攜鈣素(Calmodulin, CaM)結合導致E2的去泛素作用(Deubiquitination)及增加E2的蛋白質穩定性,由NRIP及攜鈣素所形成的複合體可以活化鈣調磷酸酶(Calcineurin, CaN)使得E2蛋白發生去磷酸化以及穩定性增加。我們呈現了E2蛋白在體內磷酸化的證據並且提出NRIP可以做為一個支架蛋白(scaffold protein)聚集E2及攜鈣素,導致E2蛋白發生去磷酸化及去泛素化進而增強蛋白質穩定性及其所驅動的基因表現。此外我們全面地評估NRIP在人類乳突瘤狀病毒16型基因表現上所扮演的角色。首先,我們發現了位於人類乳突瘤狀病毒16型啟動子上核酸7695至7710上新的質固醇受體結合位置(Glucocorticoid receptor response element, GRE);接著,我們確立了在賀爾蒙的存在下,質固醇受體調控人類乳突瘤狀病毒基因表現上NRIP可作用為轉錄輔因子;在已發現人類乳突瘤狀病毒啟動子的四個質固醇受體接合位置上,NRIP只作用於第三位置。同時在沒有賀爾蒙存在下,NRIP可與E2病毒蛋白形成一複合體結合在人類乳突瘤狀病毒啟動子上的E2結合位置(E2 response element, E2E)來調控基因表現,並且在賀爾蒙存在下,NRIP可以與質固醇受體及E2病毒蛋白形成一個三蛋白複合體作用在啟動子上的質固醇結合位置而非E2結合位置上調控基因的表現。這些結果指出NRIP與質固醇受體同為E2蛋白的結合蛋白,並且在賀爾蒙存在或不存在下,NRIP在調控人類乳突瘤狀病毒基因表現上可分別透過啟動子上的質固醇受體結合位置或E2結合位置。 We previously identified a gene, nuclear receptor-interaction protein (NRIP), which functions as a transcription cofactor in glucocorticoid receptor (GR) and human papillomavirus E2 (HPV E2)-driven gene expression. We demonstrate that NRIP is a novel binding protein for human papillomavirus 16 (HPV-16) E2 protein. HPV-16 E2 and NRIP can directly associate into a complex in vivo and in vitro, and the N-terminal domain of NRIP interacts with the transactivation domain of HPV-16 E2. Only full-length NRIP can stabilize E2 protein and induce HPV gene expression, and NRIP silenced by two designed siRNAs decreases E2 protein levels and E2-driven gene expression. We found that NRIP can directly bind with calmodulin in the presence of calcium through its IQ domain, resulting in decreased E2 ubiquitination and increased E2 protein stability. Complex formation between NRIP and calcium/calmodulin activates the phosphatase calcineurin to dephosphorylate E2 and increase E2 protein stability. We present evidences for E2 phosphorylation in vivo, and show that NRIP acts as a scaffold to recruit E2 and calcium/calmodulin to prevent polyubiquitination and degradation of E2, enhancing E2 stability and E2-driven gene expression. Moreover, we comprehensively evaluated the role of NRIP in HPV-16 gene expression. First, we identified a novel glucocorticoid response element (GRE4) located at nucleotides 7695 to 7710 of the HPV-16 promoter. We then determined that NRIP acts as a transcription cofactor to enhance GR-regulated HPV-16 gene expression in the presence of hormone, NRIP only located at the GRE3 site of the HPV promoter among four identified GRE sites. Additionally, NRIP can form complex with E2 that caused NRIP-induced HPV gene expression via E2-binding sites in a hormone-independent manner. Furthermore, NRIP can associate with GR and E2 to form tri-protein complex to activate HPV gene expression via GRE, not the E2-binding site, in a hormone-dependent manner. These results indicate that NRIP and GR are viral E2-binding proteins and that NRIP regulates HPV gene expression via GRE and/or E2 binding site in the HPV promoter in a hormone-dependent or independent manner, respectively. |
URI: | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/48118 |
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