Degradation Mechanism of the Human Placenta-Specific Transcription Factor GCMa

中文摘要 人類胎盤特殊轉錄因子hGCMa屬於GCM轉錄因子基因家族，可控制融合蛋白syncytin基因表現，促使胎盤絨毛表面的細胞滋養層細胞(cytotrophoblast)融合為融合細胞滋養(syncytiotrophoblast)，此對胎盤特化絨毛構造的形成十分重要，同時也是維持胚胎發育的必要過程。對於hGCMa的生理弁鄐w有釵h文獻進行探討，然而此轉錄因子活性的調控機制卻仍然未明。 本實驗計畫，證明hGCMa蛋白在細胞中可受長鏈polyubiquitin chain標定，並由26S proteasome進行降解，同時，和F-box protein p50存有專一性的交互作用，而F-box protein即是SCF complex這種E3中，負責識別受質的蛋白分子。進一步的研究指出，hGCMa藉由蛋白質N端的GCM motif和p50結合，且需在此轉錄因子被磷酸化後，p50才能與其產生交互作用。另外，我們也發現帶有F-box protein p50的SCFp50，可減低細胞中hGCMa蛋白的含量；而除去F-box motif的p50或F-box protein p46則無此能力。然而，除去F-box motif的p50已無法引導蛋白降解，但將其和hGCMa共同轉染，可發現此種具有缺陷的p50蛋白和野生型p50蛋白，均具有抑制hGCMa轉錄活性的能力。 上述證據顯示，轉錄因子hGCMa在細胞中受ubiquitin-proteasome蛋白降解路徑的控制，且此蛋白應是迄今第一種被鑑定出來由SCFp50這種E3引導的受質。而F-box protein p50除了引導hGCMa進行降解之外，本身也可能是此轉錄因子的協同抑制因子(co-repressor)。總括而言，本篇報告有助於釐清hGCMa的降解機制，並可協助瞭解此轉錄因子在生物體內的活性調控方式。Abstract hGCMa, a member of glial cells missing (GCM) family, is specifically expressed in human placenta. By inducing the expression of the gene encoding the fusogenic protein, syncytin, hGCMa can regulate the fusion of cytotrophoblasts into the syncytiotrophoblast layer, which is essential for the formation of chorionic villous and pregnancy maintenance. Though several studies have been made to investigate the biological functions of hGCMa, little is known about how this transcription factor is regulated. In this report, we demonstrate that hGCMa can be modified with polyubiquitin chains and degraded by the 26S proteasome in vivo. An F-box protein p50, the receptor component of the SCF E3 complex, was identified to interact with hGCMa in a phosphorylation-dependent manner. The GCM motif located at N-terminal fragment of hGCMa was found to be essential for interacting with p50. Furthermore, SCFp50 complex decreased the steady-state protein level of hGCMa in cells, but not the SCF complexes assembled with either F-box-deleted p50 or F-box protein p46. Meanwhile, cotransfection of hGCMa with F-box-deleted p50 or wild-type p50 repressed the transcriptional activity of hGCMa. These results suggest that degradation of hGCMa is regulated by the ubiquitin-proteasome pathway and hGCMa is the first SCFp50 substrate ever been identified. In addition to mediating the degradation, p50 can also serve as a transcription co-repressor of hGCMa. Overall, this study reveals the degradation mechanism of hGCMa and may help understand how its transcriptional activity is controlled in vivo.目 錄 目 錄 I 圖表目錄 III 縮寫表 IV 中文摘要 V 英文摘要 Abstract VI 第一章 緒論 1 1.1 胎盤 1 1.2 gcm轉錄因子基因家族 3 1.3 Ubiquitin-proteasome蛋白降解路徑 6 1.4 E3 – Ubiquitin接合酵素 10 1.5 研究動機及目標 12 第二章 材料及方法 13 2.1 重組質體的構築和增殖 13 2.2 重組蛋白在人類細胞的表現 17 2.3 共同免疫沉澱法 (Co-immunoprecipatation) 20 2.4 活體外交互作用分析 (In vitro “pull-down” assay) 21 2.5 螢光報告基因Luciferase活性檢測 (Luciferase assay) 22 第三章 實驗結果 23 3.1 hGCMa胺基酸序列分析 23 3.2 Ubiquitin對hGCMa的標定 24 3.3 hGCMa和SCF複合體的交互作用 25 3.4 hGCMa和F-box protein的交互作用 26 3.5 影響p50和hGCMa間交互作用的因子 28 3.6 p50和hGCMa交互作用生理意義的分析 30 第四章 討論與總結 32 4.1 hGCMa活性受ubiquitin-proteasome蛋白降解路徑調控 32 4.2 RING finger E3 – SCFp50引導hGCMa降解 33 4.3 F-box protein p50可能是hGCMa的協同抑制因子 34 4.4 影響hGCMa蛋白降解的訊號 35 4.5 胎盤轉錄因子hGCMa的降解機制模式 36 第五章 圖表 37 第六章 參考文獻 5