2012-08-012024-05-13https://scholars.lib.ntu.edu.tw/handle/123456789/643737摘要：除了在能量代謝上扮演了重要角色外，調控細胞死亡也是粒線體的重要功能，且在近年來的細胞死亡相關研究中佔有一席之地。許多導致細胞死亡的訊息傳遞路徑會影響粒線體的正常功能，例如:造成粒線體外膜的通透度增加，使一些粒線體中調控細胞死亡蛋白釋放到細胞質中，或是造成粒線體產生活性氧化物質，因而造成粒線體功能的破壞。以上的現象大都是經由位於粒線體外膜上的Bcl-2 家族蛋白來調控，並且和細胞凋亡、細胞壞死以及細胞自噬的機制都有關。Bcl-2 家族蛋白可分為抗細胞凋亡、促進細胞凋亡、以及只具有BH3 domain 的蛋白。抗細胞凋亡以及促進細胞凋亡的蛋白是以複雜的交互作用機制，來共同調控細胞死亡的路徑。然而Bcl-2 家族蛋白是否具有調節粒線體的能量生成的作用目前仍未知。RIP1 這個蛋白激脢過去被認為是活化NF-κB 路徑以抑制細胞凋亡的重要蛋白，但近來發現它也在necroptosis (一種在缺乏細胞凋亡機制的狀況下由活化的死亡受體所誘發的死亡)及parthanatos (和PARP-1 過度活化相關的死亡)的細胞壞死形式中，參與在粒線體產生活性氧化物質的過程。RIP1 也可能參與在廣效性蛋白脢抑制劑zVAD 導致小鼠纖維母細胞瘤L929 細胞自噬死亡路徑中。但是目前為止，RIP1影響粒線體功能導致細胞壞死及細胞自噬死亡的調控機制是否與Bcl-2 家族蛋白有關並沒有被研究。近來我們的實驗結果顯示，RIP1 參與在小鼠纖維母細胞中由Fas/zVAD 導致的活性氧化物質產生以及necroptosis，由DNA damager MNNG 造成的活性氧化物質產生以及parthanatos，以及在zVAD 導致小鼠纖維L929 母細胞瘤之細胞自噬性死亡。我們也發現JNK 的活化參與在Fas/zVAD 和MNNG 導致小鼠纖維母細胞活性氧化物質的增加，也參與在zVAD 引起L929 細胞自噬路徑中。因此本計畫擬延續Fas/zVAD，MNNG，zVAD 三種死亡誘導劑來闡述RIP1 和Bcl-2 家族蛋白是否在粒線體上具有交互作用，而調控細胞壞死及細胞自噬性死亡。研究目標分列六點如下：1. 研究 RIP1 和Bcl-2 家族蛋白交互作用的專一性以及結構上交互作用的區域。2. RIP1 和Bcl-2 家族蛋白交互作用上的功能性探討。3. RIP1 和Bcl-2 家族蛋白的交互作用在調控氧化性磷酸化以及粒線體能量生成的功能性探討。4. 研究 RIP1 之酵素活性是否參與Bcl-2 家族蛋白的交互作用及影響調控粒線體媒介的細胞死亡及能量生成的路徑。5. 研究調控死亡受體(Fas)、DNA 斷裂物(MNNG)以及zVAD 傳遞訊息至RIP1 而影響粒線體功能的機制。6. 研究 PARP-1 如何藉由ADP 核醣化反應造成粒線體功能的破壞，以及調控RIP1 和Bcl-2 家族蛋白的功能。<br> Abstract: In addition to the established role of the mitochondria in energy metabolism, regulation ofcell death has emerged as a second major function of these organelles, and has placed themitochondria in the focus of current cell death research. Many lethal signal transductionpathways converge on mitochondria to cause the permeabilization of the mitochondrial outermembrane (MOMP), leading to the cytosolic release of death-regulating proteins, generationof reactive oxygen species (ROS), and to the impairment of the bioenergetic functions ofmitochondria. All above parameters derived from mitochondria capable of regulatingdifferent death paradigms, e.g. apoptosis, necrosis and autophagic cell death, are controlledby mitochondrial-localizing Bcl-2 family proteins. Bcl-2 family members includeantiapoptotic, proapoptotic and BH3-only protein families. The different anti- andpro-apoptotic members of this family form a highly selective network of functionalinteraction that play a key role in mitochondria-dependent death pathways leading toapoptosis, necrosis and autophagy. Currently whether Bcl-2 family proteins also participateto regulate mitochondrial bioenergetics remains unknown. RIP1, a central component of theNF-κB signaling network leading to inhibition of cell apoptosis, also participates inmitochondrial ROS production which contributes to necroptosis (mediated by death receptoractivation under deficiency of apoptosis machinery) and parthanatos (associated withPAPR-1 overactivation) in a variety of cell types. In addition, RIP1 is involved inpan-caspase inhibitor zVAD-induced autophagic cell death in L929 fibrosarcoma. Currentlywhether RIP1 is involved in Bcl-2 family-controlled death pathways for apoptosis, necrosisand autophagic death remains undetermined. Our recent studies further demonstrate theinvolvement of RIP1 in Fas/zVAD-mediated ROS increase and necroptosis in mouseembryonic fibroblasts (MEFs), DNA damager MNNG-induced ROS production andparthanatos in MEFs, and zVAD-induced autophagic cell necrosis in L929 fibrosarcoma.Notably, we also observed JNK activation is required for the ROS increase induced by Fasand MNNG in MEFs. In addition, JNK is involved in autophagy formation induced by zVADin L929 cells. The major aim of this study is to use the three identified death models in MEFs(caused by Fas/zVAD and MNNG) and L929 cells (caused by zVAD) to further explore thelink between RIP1 and Bcl-2 family proteins in mitochondria-mediated cell necrosis andautophagic cell death. Specific aims of this project includeAim 1: To delineate the interaction specificity and binding domains between RIP1 and Bcl-2family members.Aim 2: Functional characterization and molecular basis of RIP1 in cooperation with Bcl-2family members to control cell death.Aim 3: Functional characterization and molecular basis of RIP1 in cooperation with Bcl-2family members to control oxidative phosphorylation and mitochondrialbioenergetics.Aim 4: To investigate the role of catalytic activity of RIP1 in regulating the interaction withBcl-2 family members and in mediating mitochondria-based cell death andbioenergetics.Aim 5: To delineate how death signals from plasma death receptors, DNA damage and zVADtransduce signaling cascades to initiate RIP1-mediated actions in mitochondria.Aim 6: To investigate how ADP-ribosylation caused by PARP-1 is involved in mitochondrialdysfunction and relevance to RIP1 and Bcl-2 function in mitochondria.RIP1蛋白激脢NF-kBMNNGPARP-1Bcl-2RIP1NF-kBMNNGPARP-1Bcl-2