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研究生(外文):Lan-Yu Wu
論文名稱(外文):BNIP3 Involves Endoplasmic Reticulum Stress-induced Cell Death in Murine Neuroblastoma Cells
指導教授(外文):Shye-Jye Tang
外文關鍵詞:BNIP3ER stress
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Bcl-2 nineteen-kilodalton interacting protein (BNIP3)為Bcl-2家族中調控細胞死亡因子的Bcl-2 homology domain (BH3)-only成員之一。BNIP3含有易降解的PEST domain,以及位於C端的高保守性transmembrane (TM) domain,經細胞內壓力,TM domain會促使BNIP3形成穩定的雙聚體複合物(homodimerization complexes)並與粒線體外膜結合。文獻指出,去除BNIP3 TM domain(BNIP3△TM)會降低BNIP3促進細胞死亡能力,可能是TM domain缺陷無法與粒線體外膜結合造成的結果。實驗結果發現BNIP3△TM主要以單體(monomer)形式表現,表示BNIP3的形成雙體為細胞死亡的關鍵因素。研究指出,BNIP3可被磷酸化,而磷酸化BNIP3與BNIP3誘導細胞死亡的作用有關。BNIP3會促細胞凋亡(apoptotic)或非細胞凋亡的細胞死亡路徑,如細胞自噬(autophagy)或細胞壞死(necrosis)。
本實驗利用鈣離子載體A23187處理Neuro-2a細胞導致細胞染色質濃縮(DNA condensation)、細胞自噬(Autophagy)以及粒線體失活的現象之外,同時BNIP3的表現量增加,表示BNIP3參與細胞死亡調控。研究中Neuro-2a細胞轉染 pEGFP-BNIP3處理蛋白酶抑制劑MG132,GFP-BNIP3表現量增加,表示GFP-BNIP3經由proteasome -dependent degradation pathway進行蛋白質降解。
此實驗將BNIP3序列的酪胺酸突變為丙胺酸,製備無法磷酸化的BNIP3(GFP-BNIP3 A33A92A175),或天冬胺酸,製備擬磷酸化的BNIP3(GFP-BNIP3 D33D92D175),結果表示GFP-BNIP3 DDD不形成雙體。進一步將Neuro-2a細胞轉染pEGFP-BNIP3 Y33Y92D175或pEGFP-BNIP3 D33D92Y175,結果發現GFP-BNIP3 D33D92Y175不形成雙體,顯示由Y175對於BNIP3形成雙體有重要的作用,而細胞內BNIP3大量表現會造成粒線體失活。

Bcl-2 nineteen-kilodalton interacting protein (BNIP3), the BH3-only protein, is members of the Bcl-2 family of cell death-regulating factors. The major domains found in BNIP3 contains the PEST domain that targets BNIP3 for degradation, and a conserved transmembrane (TM)domain at its C-terminus ,which targets BNIP3 to form stable homodimerization complexes and localize to the outer membrane of the mitochondria. Several studies have shown that the deletion of the TM domain (△TM) abolished the pro-death activity of the BNIP3. It is possible that this functional defect of the BNIP3△TM mutants might be the result of their inability to localize in the mitochondria. BNIP3△TM major form is monomers, that appears that the unique stable dimerization activity of BNIP3 is not critical for its cell death activity. BNIP3 was found that phosphorylation and elevation of phospho-BNIP3 resulted in apoptotic or nonapoptotic cell death, autophagy or necrosis.
In this study , Neuro-2a cells were treated with Calcium ionophore (A23187), The A23187-induced cell death was showed by DNA condensation, autophagy, mitochondria dysfunction, and the up-regulation of BNIP3 was found in Neuro-2a cells. Proteasome inhibitor, MG132, increased the stability of the protein, indicating that BNIP3 degradation is through proteasome -dependent pathway. To characterize tyrosine phosphorylation involving BNIP3-dependent cell death, tyrosines of BNIP3 were mutated to Alanine (GFP-BNIP3 A33A92A175)to form unphosphorylated BNIP3 or Aspartic acid(GFP-BNIP3 D33D92D175) to mimic tyrosine phosphorylation. After transfection BNIP3 D33D92D175 forms monomers, and the mitochondria dysfunction when BNIP3 overexpression in cells .


謝辭 I
摘要 II
英文摘要 IV
目錄 VI
圖目錄 VIII
附圖 50
壹、序論 1
3. BCL-2蛋白質家族(BCL-2 FAMILY) 2
4. BNIP3蛋白質 3
貳、研究動機 5
参、實驗材料 6
1. 細胞培養 (CELL CULTURE) 6
2. 酵素 6
3. 試劑 7
4. MAKER 7
5. DNA 8
6. 抗體 8
肆、實驗方法 11
1. 冷凍細胞活化 11
2. 細胞繼代 11
3. 計算細胞濃度 12
4. 細胞冷凍保存 12
5. 西方墨點法 ( WESTERN BLOTTING ) 12
6. 反轉錄連鎖聚合反應 (RT-PCR) 16
8. 細胞自噬(AUTOPHAGY) 19
9. 粒線體活性 19
伍、研究結果 20
陸、討論 25
柒、參考文獻 29

Ahdab-Barmada, M., J. Moossy, et al. (1986). "Hyperoxia produces neuronal necrosis in the rat." Journal of Neuropathology & Experimental Neurology 45(3): 233.

Aouacheria, A., F. Brunet, et al. (2005). "Phylogenomics of life-or-death switches in multicellular animals: Bcl-2, BH3-Only, and BNip families of apoptotic regulators." Molecular biology and evolution 22(12): 2395-2416.

Berridge, M. J., M. D. Bootman, et al. (2003). "Calcium signalling: dynamics, homeostasis and remodelling." Nature Reviews Molecular Cell Biology 4(7): 517-529.

Boyd, J. M., S. Malstrom, et al. (1994). "Adenovirus E1B 19 kDa and Bcl-2 proteins interact with a common set of cellular proteins." Cell 79(2): 341-351.

Brookes, P. S., Y. Yoon, et al. (2004). "Calcium, ATP, and ROS: a mitochondrial love-hate triangle." American Journal of Physiology-Cell Physiology 287(4): C817-C833.

Burton, T. R. and S. B. Gibson (2009). "The role of Bcl-2 family member BNIP3 in cell death and disease: NIPping at the heels of cell death." Cell Death & Differentiation 16(4): 515-523.

Burton, T. R., E. S. Henson, et al. (2006). "The pro‐cell death Bcl‐2 family member, BNIP3, is localized to the nucleus of human glial cells: Implications for glioblastoma multiforme tumor cell survival under hypoxia." International journal of cancer 118(7): 1660-1669.

Carafoli, E. (2002). "Calcium signaling: a tale for all seasons." Proceedings of the National Academy of Sciences 99(3): 1115.

Chaney, M. O., P. V. Demarco, et al. (1974). "Structure of A23187, a divalent cation ionophore." Journal of the American Chemical Society 96(6): 1932-1933.

Chinnadurai, G., S. Vijayalingam, et al. (2008). "BNIP3 subfamily BH3-only proteins: mitochondrial stress sensors in normal and pathological functions." Oncogene 27: S114-S127.
Cizeau, J., R. Ray, et al. (2000). "The C. elegans orthologue ceBNIP3 interacts with CED-9 and CED-3 but kills through a BH3-and caspase-independent mechanism." Oncogene 19(48): 5453-5463.

Gandhi, S. and A. Y. Abramov (2012). "Mechanism of Oxidative Stress in Neurodegeneration." Oxidative Medicine and Cellular Longevity 2012.

Graham, R. M., J. W. Thompson, et al. (2007). "Regulation of Bnip3 death pathways by calcium, phosphorylation, and hypoxia-reoxygenation." Antioxidants & redox signaling 9(9): 1309-1316.

Gunter, T., L. Buntinas, et al. (2000). "Mitochondrial calcium transport: mechanisms and functions." Cell calcium 28(5-6): 285.

Guntera, T. E., D. I. Yuleb, et al. (2004). "Calcium and mitochondria." FEBS letters 567(1): 96-102.

Gustafsson, Å. B. (2011). "Bnip3 as a dual regulator of mitochondrial turnover and cell death in the myocardium." Pediatric cardiology 32(3): 267-274.

Halliwell, B. (2006). "Oxidative stress and neurodegeneration: where are we now?" Journal of neurochemistry 97(6): 1634-1658.

Lemasters, J. J., A. L. Nieminen, et al. (1998). "The mitochondrial permeability transition in cell death: a common mechanism in necrosis, apoptosis and autophagy." Biochimica et biophysica acta 1366(1-2): 177.

Mellor, H. R. and A. L. Harris (2007). "The role of the hypoxia-inducible BH3-only proteins BNIP3 and BNIP3L in cancer." Cancer and Metastasis Reviews 26(3): 553-566.

Modjtahedi, N., F. Giordanetto, et al. (2006). "Apoptosis-inducing factor: vital and lethal." Trends in cell biology 16(5): 264-272.

Parekh, A. B. and J. W. Putney Jr (2005). "Store-operated calcium channels." Physiological reviews 85(2): 757-810.

Ray, R., G. Chen, et al. (2000). "BNIP3 heterodimerizes with Bcl-2/Bcl-XL and induces cell death independent of a Bcl-2 homology 3 (BH3) domain at both mitochondrial and nonmitochondrial sites." Journal of Biological Chemistry 275(2): 1439.

Reed, P. W. and H. A. Lardy (1972). "A23187: a divalent cation ionophore." Journal of Biological Chemistry 247(21): 6970.

Regula, K. M., K. Ens, et al. (2002). "Inducible expression of BNIP3 provokes mitochondrial defects and hypoxia-mediated cell death of ventricular myocytes." Circulation research 91(3): 226-231.

Szabadkai, G. and M. R. Duchen (2008). "Mitochondria: the hub of cellular Ca2+ signaling." Physiology 23(2): 84-94.

Ura, S., H. Nishina, et al. (2007). "Activation of the c-Jun N-terminal kinase pathway by MST1 is essential and sufficient for the induction of chromatin condensation during apoptosis." Molecular and cellular biology 27(15): 5514-5522.

Webster, K. A., R. M. Graham, et al. (2005). "BNip3 and signal-specific programmed death in the heart." Journal of molecular and cellular cardiology 38(1): 35-45.

Werno, C., J. Zhou, et al. (2008). "A23187, ionomycin and thapsigargin upregulate mRNA of HIF‐1α via endoplasmic reticulum stress rather than a rise in intracellular calcium." Journal of cellular physiology 215(3): 708-714.

Xu, C., B. Bailly-Maitre, et al. (2005). "Endoplasmic reticulum stress: cell life and death decisions." Journal of Clinical Investigation 115(10): 2658.

Yamazaki, M., K. Chiba, et al. (2009). "Genipin suppresses A23187-induced cytotoxicity in Neuro2a cells." Biological and Pharmaceutical Bulletin 32(6): 1043-1046.

Yasuda, M., P. Theodorakis, et al. (1998). "Adenovirus E1B-19K/BCL-2 interacting protein BNIP3 contains a BH3 domain and a mitochondrial targeting sequence." Journal of Biological Chemistry 273(20): 12415-12421.

Zhang, J. and P. A. Ney (2009). "Role of BNIP3 and NIX in cell death, autophagy, and mitophagy." Cell Death & Differentiation 16(7): 939-946.

Zmijewski, M., R. Wong, et al. (1983). "The biosynthesis of antibiotic A23187." Tetrahedron 39(8): 1255-1263.

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