肿瘤坏死因子启动RIP3依赖而RIP1非依赖细胞程序性坏死的机制研究

Necroptosis is a new kind of programmed cell death, which plays a key role in regulating embryonic development, inflammation and ischemic damage. In previous study, receptor-interacting protein 1 (RIP1) and RIP3 have been identified as two key target proteins in mediating necroptosis induced by tumor necrosis factor alpha (TNFα). In respond to TNFα treatment, RIP1 binds RIP3 to form necrosome, and mutual direct or indirect phosphorylation of RIP1 and RIP3 in the necrosome activates necrotic signaling. Depletion of RIP1 or RIP3 abrogates TNFα-induced necroptosis in many kinds of necroptosis model. Therefore, necrosome consisting of RIP1 and RIP3 becomes the essential initiator for TNFα-induced necroptosis. However, our previous study displayed that depletion of RIP1 did not inhibit TNFα-induced necroptosis in SH-SY5Y cells, but depletion of both RIP1 and RIP3 inhibited necroptosis induced by TNFα, indicating that RIP3 mediated TNFα-induced necrotic signaling through RIP1-independent manner. Recently, it is reported that phosphorylated RIP3 mediated TNFα-induced necroptosis by activating Drp1 and promoting mitochondrial fragmentation . Our previous study identified that inhibition of Drp1 activation with specific inhibitor also significantly inhibited TNFα-induced necroptosis in RIP1 depleted SH-SY5Y cells. Therefore, in respond to TNFα treatment, RIP3 may form RIP1-independent necrosome and activate to initiate necroptosis. However, it is not known which proteins were recruited by RIP3 to form RIP1-independent necrosome and the detail role of RIP3 activity in regulating RIP1-independent necroptosis induced by TNFα. The protein kinase that mediates the phosphorylation of RIP3 has not been identified, too. Therefore, in our following investigation, we will focus on the identification of new necrosome and detail mechanism in mediating RIP1-independent necroptosis induced by TNFα. The investigation would identify new target and mechanism in regulating necroptosis and provide potential target for the precaution and treatment of illness associated with necroptosis.

细胞程序性坏死（简称细胞坏死）是一种新的细胞程序性死亡，在胚胎发育、炎症反应及缺血性心脑组织损伤等生理和病理过程中都具有重要调控作用。目前的研究表明RIP1激酶与RIP3激酶结合形成坏死复合体是肿瘤坏死因子α（TNFα）诱导细胞坏死的关键环节。但我们发现在RIP1敲除的SH-SY5Y细胞中TNFα依然能够诱导细胞坏死，而同时敲除RIP1和RIP3可拮细胞抗坏死，且RIP3下游蛋白Drp1的抑制剂也可抑制细胞坏死。提示在RIP1缺失的细胞中，TNFα介导的细胞坏死信号通过RIP1非依赖的途径传递到RIP3，从而启动细胞坏死。本项目将围绕RIP3单独介导TNFα诱导的细胞坏死这一现象展开研究，寻找并鉴定将TNFα诱导细胞坏死的信号传递到RIP3的新型复合体，阐明TNFα诱导RIP1非依赖性细胞坏死的调控机制，从而进一步揭示细胞坏死的调控机制，为相关疾病的预防和治疗提供潜在靶点和理论依据。

RIP1与RIP3是调控细胞程序坏死的节点蛋白，在TNFα及其他因素诱导的细胞程序性坏死过程中，二者相互结合形成“坏死复合体”，并通过相互磷酸化或在其他蛋白激酶的作用下激活，进而活化RIP3的下游靶蛋白MLKL来启动细胞程序性坏死。但我们的前期研究发现敲低RIP1并不能够抑制TNFα单独及与Z-VAD联合诱导的L929细胞死亡，表明TNFα能够诱导RIP1非依赖的细胞程序性坏死，但其调控靶点及具体机制并不清楚。本研究发现虽然TRADD在正常的L929细胞中并不介导RIP1依赖的细胞程序性坏死，但在RIP1非依赖的细胞程序性坏死过程中却具有关键性的调控作用。此外，RIP3及其激酶活性也是调控RIP1非依赖的细胞程序性坏死的重要因素。TRADD通过调控MLKL的磷酸化及多聚化来调控TNFα诱导的RIP1非依赖的细胞程序性坏死，且对该过程中RIP3的多聚化修饰也具有重要调控作用。敲低RIP3能够促进TNFα诱导的细胞程序坏死转变为细胞凋亡，而TRADD在该过程中具有关键性的调控作用。本研究发现PI3K抑制剂及敲低PI3K的催化亚基p110能够显著抑制TNFα诱导的RIP1依赖的及非依赖的细胞程序性坏死，且PI3K的这项功能与其下游的靶蛋白PDPK1及AKT无关。因此，作为丝/苏氨酸蛋白激酶的PI3K有可能作为RIP3的蛋白激酶来调控细胞程序性坏死。本研究发现Rapamycin及敲低FKBP12都能够抑制TNFα诱导的细胞程序坏死，具体机制可能与FKBP12对RIP1的调控相关。此外，Rapamycin完全抑制佐剂诱导的大鼠类风湿性关节炎的发生与发展，揭示细胞程序性坏死可能参与自身免疫性疾病的调控。本研究鉴定了调控RIP1非依赖的细胞程序性坏死的靶蛋白与具体机制，同时明确了PI3K及FKBP12调控细胞程序性坏死的作用，完善了细胞程序性坏死的调控理论，为细胞程序性坏死相关疾病的临床治疗及药物设计提供了新的靶点与理论参考。