楊性芳臺灣大學：分子醫學研究所劉軒Liu, HsuanHsuanLiu2007-11-262018-07-092007-11-262018-07-092005http://ntur.lib.ntu.edu.tw//handle/246246/51370Mcl-1 屬於Bcl-2 家族之一，其功能在發育過程中扮演了重要的角色。Mcl-1之極短半衰期及其蛋白質含量為許多細胞存活路徑之重要調控者，因此我們推測，細胞內調控Mcl-1蛋白的穩定性以及調控其合成的機制對於Mcl-1的功能同等重要。細胞受到許多生理刺激均會引發Mcl-1蛋白質含量之增加，此過程在轉錄機制方面有許多詳細的研究，但對於其穩定性的調控機制卻不是很清楚。此篇論文中，我們發現Translationally Controlled Tumor Protein (TCTP)會和Mcl-1作用而進一步調控Mcl-1蛋白的穩定性。當細胞內大量表現TCTP可以增加Mcl-1的半衰期，相反的，利用RNA干擾的方式來降低TCTP的表現則會降低Mcl-1蛋白的穩定性。研究TCTP基因愓除的老鼠則發現，Mcl-1的含量在TCTP+/-的老鼠中明顯的比TCTP+/+的低。我們的結果顯示，TCTP主要是藉由抑制Mcl-1的ubiqutinalation而影響Mcl-1的分解，因此而增加蛋白的半衰期。研究在TCTP結合上有缺失的Mcl-1突變蛋白(K257V)則發現其分解的速度加快, 且抗細胞凋亡的能力也相對降低。因此，我們推測，TCTP透過調控Mcl-1蛋白的穩定性，而影響其抗細胞凋亡的能力。其中可能牽涉之作用機轉，將在此論文中討論。 此外，我們的結果也顯示，生長訊號會同時調控Mcl-1和TCTP的蛋白表現量和細胞內的位置。 當細胞處於去除血清的情況之下，二者蛋白質都會在核中出現，而再使用血清刺激細胞時，二者則都會位於細胞質中。我們初步的結果顯示，TCTP可能會調控Mcl-1在細胞中的的位置，但此種現象對Mcl-1功能上的重要性並不清楚，需待更進一步的研究。Mcl-1 is one Bcl-2 family member that plays a pivotal role in animal development. The extremely labile nature of the Mcl-1 protein itself and the fact that the Mcl-1 level is a critical determinant in various cell survival pathways suggest that cellular processes that regulate Mcl-1 stability are as important as those regulate Mcl-1 synthesis. While transcriptional stimulation of Mcl-1 synthesis in response to various stimuli has been well documented, regulation of Mcl-1 stability has been hardly explored. In this study, we identified that the Translationally Controlled Tumor Protein (TCTP) was one cellular factor that interacted with Mcl-1 and modulated Mcl-1 stability. While over-expression of TCTP augmented the protein stability of Mcl-1, knockdown expression of TCTP by RNA interference destabilized Mcl-1. Consistently, targeted ablation of the TCTP gene led to a reduced level of Mcl-1. Furthermore, TCTP stabilized Mcl-1 through interfering with Mcl-1’s degradation by the ubiquitin-dependent proteasome degradation pathway, and the TCTP binding-defective mutant of Mcl-1 (K257V) was much more susceptible to degradation and manifested a compromised anti-apoptotic activity. Taken together, these results suggest that TCTP modulates Mcl-1’s anti-apoptotic activity by modulating its protein stability. The possible mechanism(s) involved in TCTP’s modulation process is discussed. Additionally, our results demonstrated an involvement of growth signaling in modulating the expression and subcellular localization of Mcl-1 and TCTP. Both proteins, which generally locate in the cytosol of rapidly growing cells, could be detected in the nuclear compartment when cells were deprived of serum. Furthermore, interaction with TCTP may be an important determinant in regulating the localization of Mcl-1. However, the functional significance of the nuclear localization of these proteins is not fully understood and awaits further investigation.中文摘要………………………………………………….……….……….………………….4 Abstract……...…………………………………….…...……………….…….…………….…5 Introduction………………………………………...………….………………….................6 Material and method Yeast Two-Hybrid Screening………………………………………………….…………………21 Antibodies…………………………………………………………………………......................21 Cell culture and generation of stable cell lines…………….…………………….………………22 Expression vectors……………………………………………………………………………….23 In vitro GST pull-down assay …………………………………………………….……………..25 Co-immunoprecipitation of TCTP and mMcl-1…………….………………….…......................25 Binding of endogenous mouse Mc-l and TCTP…………….………………...……....................26 Indirect mmunofluorescence and confocal laser scanning microscopy.………….………..…….27 Determination of Mcl-1 protein half-life…………….…………………………….…………….27 Determination of Mcl-1 synthesis rate…………….…………………………..…………………28 In vivo ubiquitination of mMcl-1………………………………………………..……………....29 Knockdown of TCTP expression with small interfering RNA (siRNA)..………….....................29 Results Interaction between Mcl-1 and TCTP…………….……………………………….……………..31 TCTP enhances the protein stability of Mcl-1 but not vice versa…………………......................33 Knockdown expression of TCTP destabilizes Mcl-1…………….……………............................35 The TCTP binding-defective Mcl-1 mutant is much less stable and manifests a compromised anti-apoptotic activity …………….……………………………….……………….……………36 TCTP blocks ubiquitination of Mcl-1…………….……………………………….......................37 Mcl-1 expression level is altered in TCTP+/- mice…………….………………….…………..…38 The expression and subcellular localization of Mcl-1 and TCTP are both regulated in response to serum stimulation in the mouse NIH3T3 fibroblasts and human HeLa cells…………….….......39 The faster-migrating form of Mcl-1 is localized in the nucleus…………………...……….……40 Interaction with TCTP may be an important determinant in Mcl-1’s subcellular localization…..41 Microtubule-association of Mcl-1 and TCTP in mitotic cells…………….………......................41 Discussion…………….………………………………………….…………………………..43 Figures…………….……………………………….………………………...…………...…..49 References…………….……………………………………………………………………..72 Appendix 1: notification of acceptance…………………………..…..…………81 Appendix 2: submitted manuscript………………………………..…834349653 bytesapplication/pdfen-US訊號傳遞細胞凋亡signal transductionapoptosisotherhttp://ntur.lib.ntu.edu.tw/bitstream/246246/51370/1/ntu-94-D88448004-1.pdf