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

陳姵君(Pei-Chun Chen)

指導教授 ： 葉添順

臺北醫學大學/醫學院/細胞及分子生物研究所/碩士(2002年)

摘要

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。

關鍵字

酵母菌雙雜交系統

並列摘要

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.

並列關鍵字

Notch ； Yeast Two-hybrid

參考文獻

Artavanis-Tsakonas, S., M. D. Rand, and R. J. Lake. (1999) Notch signaling: cell fate control and signal integration in development. Science 284: 770-776.

Brown, M. S., J. Ye, R. B. Rawson, and J. L. Goldstein. (2000) Regulated intramembrane proteolysis: a control mechanism conserved from bacteria to humans. Cell 100: 391-398.

Capobianco, A. J., P. Zagouras, C. M. Blaumueller, S. Artavanis-Tsakonas, and J. M. Bishop. (1997) Neoplastic transformation by truncated alleles of human NOTCH1/TAN1 and NOTCH2. Mol. Cell. Biol. 17: 6265-6273.

Ellisen, L. W., J. Bird, D. C. West, A. L. Soreng, T. C. Reynolds, S. D. Smith, and J. Sklar. (1991) TAN-1, the human homolog of the Drosophila Notch gene, is broken by chromosomal translocations in T lymphoblastic neo-plasms. Cell 66: 649-661.

Fortini, M. E., and S. Artavanis-Tsakonas. (1994) The suppressor of hairless protein participates in notch receptor signaling. Cell 79: 273-282.