肿瘤坏死因子引起细胞坏死的信号通路研究

程序性细胞死亡可分为凋亡和程序性坏死。与凋亡相似，程序性细胞坏死也是发育和多种器官内稳态维持的重要助力。坏死与多种病理学进程相关，如参与如心肌坏死和动脉粥样硬化等疾病。RIP3是与细胞程序性坏死密切相关的一个关键信号，其下游调节分子包括GLUL、PYGL、GLUD1、MLKL、及PGAM5。为了进一步探究TNF介导的细胞程序性坏死的机制，我们采用串联免疫沉淀纯化坏死复合体，寻找复合体的未知组分；利用定量磷酸化质谱探究RIP3的下游底物；并已经证明这两种方法的有效性。我们得到了30多个候选基因，将在细胞中敲除这些候选基因进而有效鉴定它们是否真正参与此坏死通路。对已证实与坏死相关的新基因，我们将深入探索其功能，并阐明新基因与已知信号分子如RIP3和MLKL的关系，研究它们在细胞程序性坏死通路下游引起的事件。这项研究不仅能发现程序性细胞坏死的新机制，也能为介入治疗坏死相关疾病提供新思路。

Programed cell death can be divided into apoptosis and programmed necrosis (necroptosis). Similar to apoptosis, necroptosis is also a major sculpting force in the development and homeostasis of multicellular organisms. Abnormality of this process is associated with many pathological processes and contributes to multiple human diseases such as myocardial necrosis and atherosclerosis. In the elucidation of the signaling cascade of necroptosis, receptor interacting protein kinase 3 (RIP3) has been recognized as a key signaling mediator that is intimately involved in necroptosis. Although the delineation of the signaling cascades downstream of the necrosome has revealed the involvement of GLUL, PYGL, GLUD1, MLKL, and PGAM5, the mechanisms of necroptosis still remain elusive. To further investigate the mechanisms of TNF-induced necroptosis, we employed sequential immunoprecipitation (IP) to purify the necrosome complex for identification of new necrosome components and used quantitative phosphoproteomics to find substrates of RIP3 in necroptosis. These methods have been validated to be effective in detecting already known components of the necroptotic pathway in our laboratory. As a result of these approaches, we have more than 30 uncharacterized candidate proteins that might participate in the TNF-induced necroptotic process. Genetic deletion of these candidate genes in culture cells will be conducted to determine whether a given candidate plays a role in TNF-induced necroptosis. Molecular and cell biology approaches will be employed to characterize the function of the genes that have been proven to be important for TNF-induced necroptosis. We will determine the relationship between the newly identified gene products with the known signaling molecules such as RIP3 and MLKL and explore the downstream events of the necrosome in the execution of cell death. This study should not only provide new mechanistic insight into programmed necrosis but also new ideas for therapeutic intervention for necrosis associated diseases.