指導教授：柯逢春臺灣大學：分子與細胞生物學研究所顏宏家Yan, Hong-JiaHong-JiaYan2014-11-252018-07-062014-11-252018-07-062014http://ntur.lib.ntu.edu.tw//handle/246246/261084一般而言，我們對Bcl-2 family protein的認知都是它們anti-apoptosis或是pro-apoptosis的功能，會因為彼此的交互作用，調控粒線體外膜上voltage-dependent anion channel(VDAC)的通透性，決定細胞的生死。但是近年來的研究逐漸注意到Bcl-2 family protein的另外一面，除了調控apoptosis以外，Bcl-2 family protein也影響很多細胞重要的代謝作用。 BAD是Bcl-2 family的一員，屬於pro-apoptotic的BH3-only protein，會與粒線體外膜上的Bcl-2, Bcl-xL結合，造成VDAC的通透性增加，BAD會在粒線體外膜上形成BAD complex，BAD complex會調控肝細胞glucokinase活性和beta-cells 的insulin secretion。暗示我們BAD可能在細胞的營養代謝層面上有所調控。而生長中細胞的營養可得性由粒線體anaplerosis整合。為了探討BAD活性與anaplerosis的關聯性，我們對細胞處理anaplerosis抑制劑Aminooxyacetic acid(AOA)，發現BAD的磷酸化狀態改變, p-BAD(S136)上升，p-BAD(S155)下降; 對細胞處理glutaminolysis inhibitor，BPTES也得到相同的實驗結果，證明BAD的磷酸化狀態反應粒線體anaplerosis活性。而當我們處理soluble adenylyl cyclase inhibitor, KH7，BAD磷酸化狀態的改變與抑制anaplerosis相同，說明粒線體anaplerosis活性可能經由cAMP-PKA signaling在BAD complex進行整合。此外AOA，BPTES，KH7處理皆觀察到p-S6K1(T389)下降，p-Akt(S473)上升的現象，分別代表mTORC1活性下降，mTORC2活性上升，mTORC2對Akt(S473)的活化發生於membrane raft，會進而活化p-BAD(S136)，可能借此調控BAD complex中的WAVE-1，造成粒線體到細胞膜nutrient transporter附近獲取胺基酸，促進粒線體進行anaplerosis然後產生cAMP-PKA訊號來活化p-BAD(S155)。根據以上實驗結果，證明BAD 的磷酸化狀態會反應粒線體anaplerosis活性，其中可能是由BAD complex來整合cAMP-PKA signaling，然後造成mTORC1和mTORC2活性受到影響，藉此調控細胞的營養代謝與生長。The Bcl-2 family is the best characterized protein family involved in the regulation of apoptotic cell death, consisting of anti-apoptotic and pro-apoptotic members. These proteins determine the life or dead of cells by altering the VDAC permeability, which is located on the mitochondrial outer membrane. However, recent studies suggest that bcl-2 family proteins have cellular functions beyond regulation of apoptosis. The BAD protein is a pro-apoptotic member of Bcl-2 family whose ability to heterodimerize with Bcl-2 and Bcl-xL, and increase permeability of VDAC. On the mitochondrial outer membrane, Bad assemble a complex together with PKA, PP1c, WAVE-1, and glucokinase. BAD complex and the phosphorylation state of BAD regulate the glucokinase activity in hepatocytes and the insulin secretion in beta cells, raising the possibility that BAD may be involved in nutrient metabolism. The mitochondrial anaplerosis activity is in response to cellular nutrient availability. To investigate the interaction between BAD activity and anaplerosis, we treated U2OS cells with anaplerosis inhibitor, aminooxyacetic acid(AOA), and measured the phosphorylation levels of BAD. We found that AOA induces BAD(S136) phosphorylation and reduces BAD(S155) phosphorylation. The treatments with BPTES result in the same BAD phosphorylation state, suggest that BAD phosphorylation states correspond to mitochondrial anaplerosis activity. In addition, soluble adenylyl cyclase inhibitor, KH7 treatments have the same effect. Suggest that mitochondrial anaplerosis activity may be coordinated by BAD complex through the cAMP/PKA signaling. Treatment with AOA, BPTES, KH7, both reduce the activity of mTORC1(p-S6K1-T389 decrease),and induce the activity of mTORC2(p-Akt-S473 decrease). mTORC2-Akt(S474) phosphorylates BAD at S136, may occur in membrane raft. The phosphorylation of BAD(S136)may regulate WAVE-1(the component of BAD complex), and make mitochondria move to plama membrane in order to acquire nutrient. Sequentially, cAMP-PKA signaling phosphorylate BAD(S155). The results suggest that BAD phosphorylation states correspond to mitochondrial anaplerosis activity, and mitochondrial anaplerosis activity may be coordinated by BAD complex through the cAMP/PKA signaling; furthermore affect mTORC1 and mTORC2 activity to regulate metabolism and cell growth.致謝 i 中文摘要 ii 英文摘要 iii 圖目錄 vii Chapter 1 引言 1 1.1 BAD complex參與粒線體相關的代謝作用 3 1.2 粒線體在細胞生長時的角色轉變 4 1.3 糖解作用成為細胞生長時的主要能量來源：Warburg effect 5 1.4 粒線體TCA cycle轉為生合成角色 6 1.5 細胞生長的中心調節者: mTOR complexes 7 1.6 粒線體代謝影響mTOR complexes的活性 9 1.7 cAMP-PKA signaling可能是粒線體的代謝訊息 10 Chapter 2 實驗目的 11 Chapter 3 材料與方法 12 3.1 材料 12 3.2 細胞培養 12 3.3 細胞繼代 12 3.4 細胞內含物萃取 12 3.5 蛋白質濃度測定與樣本製備 13 3.6 西方墨點法 13 Chapter 4 結果 14 4.1 AOA抑制粒線體anaplerosis使得p-BAD（Ser112）下降，p-BAD（Ser136）上升、p-BAD（Ser155）下降 14 4.2 BPTES抑制粒線體anaplerosis使得p-BAD（Ser112）上升、p-BAD（Ser136）上升、p-BAD（Ser155）下降 15 4.3 α-ketoglutarate可以回復AOA對mTORCs與BAD Ser136磷酸化的影響 15 4.4 Pyruvate可以回復AOA對mTORCs與BAD（Ser112, Ser136）磷酸化的影響 16 4.5 抑制soluble adenylyl cyclase影響mTORCs活性和BAD磷酸化狀態 16 4.6 抑制PKA使得mTORCs的活性皆上升和BAD磷酸化狀態的改變 17 Chapter 5 討論 18 5.1 粒線體代謝活性（anaplerosis/cataplerosis）對調控細胞生長的重要性 18 5.2 mTORC2-akt（Ser473）活性上升的細胞學意義 18 5.3 p-BAD（Ser136），p-BAD（Ser155）的細胞學意義 19 Reference 29834045 bytesapplication/pdf論文公開時間：2019/03/21論文使用權限：同意無償授權粒線體BADBAD complexVDACAktcAMP-PKA signalinganaplerosisAOABPTESKH7thesishttp://ntur.lib.ntu.edu.tw/bitstream/246246/261084/1/ntu-103-R99b43021-1.pdf