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Please use this identifier to cite or link to this item: http://ntour.ntou.edu.tw:8080/ir/handle/987654321/30216

Title: 一氧化氮透過BNIP3造成神經細胞的死亡,而BNIP3的酪胺酸磷酸化會降低一氧化氮造成的神經細胞死亡
Tyrosine Phosphorylated BNIP3 Decreases Nitric Oxide-induced Cell Death in Nerve Cells
Authors: Lih-Rou Rau
饒栗柔
Contributors: NTOU:Institute of Bioscience and Biotechnology
國立臺灣海洋大學:生物科技研究所
Keywords: 一氧化氮;粒線體;酪胺酸磷酸化;BNIP3
Nitric oxide;mitochondria;tyrosine phosphorylation;BNIP3
Date: 2011
Issue Date: 2011-11-25T07:36:27Z
Abstract: 中樞神經系統中,神經退化性疾病的患者中發現,氧化壓力會造成神經細胞的退化。當神經膠細胞受到免疫刺激時,會釋放出一氧化氮 。在神經膠細胞及小腸上皮細胞中發現一氧化氮的出現會使BNIP3的表現量增加,BNIP3為Bcl-2家族中具有促細胞死亡活性的BH3-only成員之一,其C端含有transmembrane (TM) domain。藉由 TM domain嵌入粒線體上,以調控細胞死亡。大部分Bcl-2家族受到轉譯後修飾的調控,BH3-only的成員多為藉由磷酸化調控其活性。BNIP3已證實可被磷酸化,而其雙體及單體的磷酸化與BNIP3造成的細胞死亡相關。 本論文實驗利用SNAP做為一氧化氮的提供者,AG490為酪胺酸激酶的抑制劑,結果發現一氧化氮造成的Neuro-2a (N2a) 細胞死亡是透過BNIP3的酪胺酸磷酸化來調控。利用MTT及Celltiter-Glo assay分析細胞存活率,發現當抑制酪胺酸激酶的活性時,會促進一氧化氮透過BNIP3造成caspase-independent途徑的細胞死亡。粒線體染色分析發現一氧化氮造成N2a細胞粒線體的聚集及喪失膜電位,同時利用西方點墨法發現也會有細胞自噬發生。 N2a細胞轉染模擬BNIP3持續磷酸化的蛋白BNIP3- Y175D, Y92D, Y33D (BNIP3-DDD) 時發現抑制酪胺酸激酶後,一氧化氮所造成的粒線體的聚集情況消失,利用免疫沉澱法及西方點墨法分析發現在N2a細胞內BNIP3-DDD表現量較低,的結果顯示BNIP3-DDD是受到蛋白質降解酶 (proteasome) 的降解,使其表現量降低。本實驗證實抑制酪胺酸激酶促進一氧化氮造成的細胞死亡為透過BNIP3造成caspase-independent途徑的粒線體失活及細胞自噬,而BNIP3會透過酪胺酸磷酸化被蛋白質降解酶所分解,以降低其造成細胞死亡的活性。
In central nervous system, oxidative stress may contribute to the neuronal degeneration in neurodegenerative diseases. Microglia activated by inflammatory stimulation may release nitric oxide (NO), which induces BNIP3 (Bcl-2/adenovirus E1B 19-kD interaction protein 3) expression observed in macrophages and enterocytes. BNIP3 is a pro-cell-death Bcl-2 family protein which contains a transmembrane domain at C terminal of BNIP3. Accroding to previous research, the majority of Bcl-2 family members are regulated post-translationally, such as phosphorylation, and BNIP3 is indeed regulated by phosphorylation on the monomeric and dimeric form, this modification are related to BNIP3 induced cell death. In this study, SNAP, a NO donor, and AG490, a tyrosine kinase inhibitor, we were used to treat Neuro-2a (N2a) cells. We found that NO- induced Neuron-2a (N2a) cell death was regulated by BNIP3, and AG490 promoted the cell death. The NO induced cell death was through mitochondria dysfunction and autophagy. To investigate tyrosine phosphorylation of BNIP3, we constructed a mutant that mimic tyrosine phosphorylation of BNIP3 (BNIP3- Y175D, Y92D, Y33D;BNIP3-DDD). N2a cell transfected with BNIP3-DDD reduced NO-induced mito- chondria aggregation. The expression of BNIP3-DDD decreased compared with the cells transfected with BNIP3. We found that proteasome may degrade tyrosine phoshprylated BNIP3 and decrease the pro-cell-death activity of BNIP3.
URI: http://ethesys.lib.ntou.edu.tw/cdrfb3/record/#G0M98360003
http://ntour.ntou.edu.tw/handle/987654321/30216
Appears in Collections:[生命科學暨生物科技學系] 博碩士論文

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