Notch1 受體活化時參與訊息傳遞的細胞因子之探討Identification of Cellular Proteins Targeted by the Activated

Notch1 Receptor

Notch 是個很大、而且穿過細胞膜一次的蛋白，最早在果蠅中被發現。哺乳類的 Notch 基因分為 Notch1?4 ，位於細胞表面，被認為可調控基因的轉錄、細胞的增生、分化及早期細胞命運的決定。當 ligand 結合在 Notch 受體的細胞外區域時， Notch 訊號傳遞系統就被活化，使得整個細胞內區域在靠近內膜處斷裂，而形成只具細胞內區域的活化型 Notch1 (Notch1 IC) 。此活化型 Notch1 會轉移至細胞核中，並且和一些可與 DNA 結合的蛋白，如： RBP-Jκ/CBF1 ，以及其他細胞因子結合，形成一個高達約 1.5 MDa 的蛋白複合體，此蛋白複合體可進一步調控其標的基因的表現。由於這些參與蛋白複合體的因子，對訊號傳遞路徑影響甚巨。因此，為了探討 Notch 訊號傳遞路徑，本論文利用 GST pull-down assay 配合蛋白體技術，尋找與 hNotch1 受體有結合作用之蛋白。並藉此發現了一個屬於人類的 glycosyltransferase 家族的蛋白： WUGSC ，可能參與 Notch 蛋白的修飾作用。另外，也利用 yeast two-hybrid 的方法，篩選在人類睪丸基因庫中會與 hNotch1-ANKΔ 結合的蛋白。結果得到六個可能與 hNotch1 有結合作用的蛋白，其中包括 ubiquitin 及 β-tubulin 。同時，也進一步利用 GST pull-down assay 及共同免疫沉澱法，證明 hNotch1 與 β-tubulin 的結合。然而， hNotch1 與 β-tubulin的結合，究竟有何意義仍須更進一步釐清。

Notch is a large, Type I transmembrane protein and identified initially in Drosophila. There are four known mammalian Notch genes, Notch1~4, that function both as receptors of cell surface and regulators of gene transcription. human Notch1 has been demonstrated to participate in the regulation of cell proliferation, differentiation, and cell fate decisions. Ligand binding initiates the signal through the proteolysis of Notch1 receptor to generate the Notch1 intracellular domain (Notch1-IC). The activated Notch1 translocates into the nucleus and associates with the DNA binding protein RBP-Jκ/CBF1 and other cofactors. The high-molecular weight complexes modulate the expression of target genes.The cellular factors of these complexes play important roles in Notch signaling pathway. In order to investigate the Notch signaling, the in vitro GST pull-down assay combined with proteomic approach were used to identify proteins interacting with hNotch1-IC. There was a candidate of hNotch1-IC associating protein, WUGSC, which is one of the glycosyltransferase family involve in modification of Notch receptor. Alternatively, used the yeast two-hybrid system to screen hNotch1-IC-ANKΔbinding proteins from human testis cDNA library. There were six candidates of hNotch1-IC associated proteins including ubiquitin and β-tubulin, identified by this approach. Futhermore, the hNotch1-IC was demonstrated to interact withβ-tubulin by GST pull-down assay and co-immunoprecipitation. The function of this interaction between hNotch1 and β-tubulin still need to be elucided.