Molecular and chemical regulations of necrotic cell death pathways

项目总结 坏死性下垂是一种坏死性细胞死亡的程序性形式，它与多种人类 疾病，包括病毒和细菌感染、炎症、缺血组织损伤以及败血症。脓毒症 是导致危重病人死亡的主要原因，每年有超过25.8万人死于 美国。这项建议的总体目标是剖析坏死性上睑下垂的分子机制，以及 开发可能有助于治疗脓毒症和其他坏死性下垂相关疾病的新型坏死性下垂抑制剂。 坏死性下垂的定义分子是受体相互作用蛋白激酶3(RIP3)，它结合其 激活时关闭同源RIP1。我们之前已经确定了一种治疗坏死性下垂的小分子抑制剂。 命名为坏死磺胺，并确定其靶点为混合谱系激酶域样蛋白(MLKL)。 激活的RIP1/RIP3招募MLKL形成死体。磷酸化的MLKL然后同源齐聚和 移位到细胞膜隔间并触发细胞死亡。然而，MLKL膜如何易位 是受监管的，细胞死亡是如何执行的仍不清楚。 我们从2,645个NCI化合物集合中进行筛选，确定了12个可以阻止下游坏死性下垂的匹配 MLKL的成员。其中，与HSP70结合的化合物NBC1和化合物NSC302979是已知的 脱泛素酶(DUB)抑制剂。我们将描述HSP70和泛素化在调节MLKL中的作用 功能和坏死性下垂。NBC1和NSC302979也将在脓毒症的小鼠模型上进行测试。我们也 进行了CRISPR-Cas9介导的全基因组敲除筛查，并确定了 坏死性下垂。确定这些基因的作用将有助于更好地理解这一途径。 此外，通过串联免疫沉淀，我们鉴定了一个MLKL相互作用蛋白Alix，它是 参与将泛素化的蛋白质运输到溶酶体内。我们还观察到溶酶体的通透性 在死后下垂期间。我们假设Alix将泛素化的MLKL招募到溶酶体中 复合体使溶酶体通透性，释放溶酶体蛋白水解酶，并执行细胞死亡。牵涉到 在坏死性下垂的执行中，溶酶体通透性将是该领域的重大概念性进展。 总之，我们的研究结合了化学生物学、生物化学和遗传学来破译 坏死性下垂的发病机制。我们目标的完成将为脓毒症的治疗和治疗提供新的化合物先导 其他与坏死性下垂相关的疾病，也将在该途径中发现新的参与者并提供更好的 对潜在机制的理解。

Project summary Necroptosis is a programmed form of necrotic cell death, which has been implicated in a variety of human diseases, including viral and bacterial infection, inflammation, ischemic tissue injuries as well as sepsis. Sepsis is the leading cause of mortality in critically ill patients and kills more than 258,000 people each year in the United States. The overall goal of this proposal is to dissect the molecular mechanisms of necroptosis, and develop novel necroptosis inhibitors that may help treat sepsis and other necroptosis-associated diseases. The defining molecular player for necroptosis is Receptor Interacting Protein Kinase 3 (RIP3), which binds its close homolog RIP1 upon activation. We have previously identified a small molecule inhibitor of necroptosis called necrosulfonamide and determined its target as Mixed lineage kinase domain-like protein (MLKL). Activated RIP1/RIP3 recruits MLKL to form necrosome. Phosphorylated MLKL then homo-oligomerizes and translocates to membrane compartments and triggers cell death. However, how MLKL membrane translocation is regulated and how cell death is executed is still not clear. We screened from 2,645 NCI compound collections and identified 12 hits that blocked necroptosis downstream of MLKL. Among them, compound NBC1 bound to HSP70 and compound NSC302979 is a known deubiquitinase (DUB) inhibitor. We will characterize the role of HSP70 and ubiquitination in regulating MLKL function and necroptosis. Both NBC1 and NSC302979 will also be tested in a mouse model of sepsis. We also conducted a CRISPR-Cas9 mediated whole genome knockout screen and identified list of genes required for necroptosis. Characterizing the role of these genes will lead to a better understanding of the pathway. Furthermore, using tandem immune-precipitation, we identified a MLKL interacting protein as Alix, which is involved in transporting ubiquitinated proteins to the lysosome. We also observed lysosome permeabilization during necroptosis. We hypothesize that Alix recruits ubiquitinated MLKL to the lysosome where MLKL complex permeabilizes lysosome and release lysosomal proteases and execute cell death. Involvement of lysosome permeabilization in necroptosis execution will be a significant conceptual advance in the field. In summary, our study uses a combination of chemical biology, biochemistry and genetics to decipher the mechanism of necroptosis. Completion of our aims will provide novel compound leads for treating sepsis and other necroptosis-associated disease, and will also identify new players in the pathway and provide better understanding of the underlying mechanisms.