Molecular and chemical regulations of necrotic cell death pathways

坏死细胞死亡途径的分子和化学调控

• 批准号: 10474826
• 负责人: Zhigao Wang
• 金额: $ 30.9万
• 依托单位: UNIVERSITY OF SOUTH FLORIDA
• 依托单位国家: 美国
• 财政年份: 2017
• 资助国家: 美国
• 起止时间: 2017-07-01 至 2023-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10474826
• 关键词: Molecular; chemical; regulations; necrotic; cell

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.

项目概要 坏死性凋亡是坏死性细胞死亡的一种程序性形式，与多种人类疾病有关。 疾病，包括病毒和细菌感染、炎症、缺血性组织损伤以及败血症。败血症 是导致重症患者死亡的主要原因，每年导致超过 258,000 人死亡 美国。该提案的总体目标是剖析坏死性凋亡的分子机制，以及 开发新型坏死性凋亡抑制剂，可能有助于治疗败血症和其他坏死性凋亡相关疾病。 坏死性凋亡的决定性分子是受体相互作用蛋白激酶 3 (RIP3)，它与其结合 激活后关闭同系物 RIP1。我们之前已经鉴定出一种坏死性凋亡的小分子抑制剂 称为死磺酰胺，并将其目标确定为混合谱系激酶结构域样蛋白（MLKL）。 激活的 RIP1/RIP3 招募 MLKL 形成坏死体。磷酸化的 MLKL 然后同源寡聚化并 易位至膜区室并引发细胞死亡。然而，MLKL膜转位如何 受到调控，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，并鉴定出 12 个可阻断下游坏死性凋亡的化合物 MLKL 的。其中，结合HSP70的化合物NBC1和化合物NSC302979是已知的 去泛素酶（DUB）抑制剂。我们将描述 HSP70 和泛素化在调节 MLKL 中的作用 功能和坏死性凋亡。 NBC1 和 NSC302979 也将在脓毒症小鼠模型中进行测试。我们也 进行了 CRISPR-Cas9 介导的全基因组敲除筛选，并确定了所需的基因列表 坏死性凋亡。表征这些基因的作用将有助于更好地理解该途径。 此外，使用串联免疫沉淀，我们鉴定了一个 MLKL 相互作用蛋白 Alix，它是 参与将泛素化蛋白质转运至溶酶体。我们还观察到溶酶体透化 坏死性凋亡期间。我们假设 Alix 将泛素化的 MLKL 募集至溶酶体，其中 MLKL 复合物透化溶酶体并释放溶酶体蛋白酶并执行细胞死亡。参与 坏死性凋亡执​​行中的溶酶体透化将是该领域的重大概念进步。 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 坏死性凋亡的机制。完成我们的目标将为治疗败血症和 其他坏死性凋亡相关疾病，还将确定该途径中的新参与者并提供更好的 了解基本机制。

项目成果

RIPK1 dephosphorylation and kinase activation by PPP1R3G/PP1γ promote apoptosis and necroptosis.

• DOI: 10.1038/s41467-021-27367-5
• 发表时间: 2021-12-03
• 期刊: Nature communications
• 影响因子: 16.6
• 作者: Du J;Xiang Y;Liu H;Liu S;Kumar A;Xing C;Wang Z
• 通讯作者: Wang Z

MLKL polymerization-induced lysosomal membrane permeabilization promotes necroptosis.

• DOI: 10.1038/s41418-023-01237-7
• 发表时间: 2024-01
• 期刊: CELL DEATH AND DIFFERENTIATION
• 影响因子: 12.4
• 作者: Liu, Shuzhen;Perez, Preston;Sun, Xue;Chen, Ken;Fatirkhorani, Rojin;Mammadova, Jamila;Wang, Zhigao
• 通讯作者: Wang, Zhigao