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Mechanisms of non-apoptotic programmed cell death and corpse clearance

非凋亡性程序性细胞死亡和尸体清除的机制

基本信息

批准号：
10292225
负责人：
Ann M Wehman
金额：
$ 43.61万
依托单位：
UNIVERSITY OF DENVER (COLORADO SEMINARY)
依托单位国家：
美国
项目类别：
财政年份：
2021
资助国家：
美国
起止时间：
2021-08-01 至 2024-07-31
项目状态：
已结题

项目摘要

Mechanisms of non-apoptotic programmed cell death and corpse clearance Project Summary/Abstract Many cells are programmed to die during development and homeostasis, but not all cells die via apoptosis. Additionally, cancer cells have proven resistant to apoptosis-promoting therapies. Therefore, it is important to understand non-apoptotic mechanisms of cell death, as well as how dying cells signal for clearance. Furthermore, corpse clearance and phagolysosomal degradation are important to avoid inflammation and auto- immune disease, but there are major gaps in our understanding of all these processes. We established that C. elegans polar bodies are non-apoptotic cells that undergo an unknown form of programmed necrosis(Fazeli et al., Cell Rep 2018)1. Using this genetic model, we propose to identify the mechanisms that lead to necrotic cell death and loss of membrane integrity in Aim 1, focusing initially on non- apoptotic caspases and scramblase regulators based on preliminary data generated by an undergraduate student. These targeted screens will identify whether polar body death shares common mechanisms with established modes of programmed necrosis or defines an undiscovered type of cell death, which may give insight into uncharacterized cell death modalities in humans during health and disease. We found that polar bodies externalize phosphatidylserine (PS) and are cleared by embryonic cells using LC3-associated phagocytosis1. However, the signaling pathways that regulate PS exposure in necrotic cells are not known. In preliminary data from an undergraduate student, we have identified at least four redundant lipid scramblases that are required for PS exposure and have reduced PS exposure by mutating them all. This approach is one of several in Aim 2 that will allow us to define the signaling pathways that regulate engulfment of the corpse as well as the recruitment of Atg8/LC3 family proteins to the phagosome. These signaling pathways may be conserved in other types of cell death, including apoptosis, providing wide-ranging insights into the mechanisms of corpse clearance. We also discovered the first role for Atg8/LC3 recruitment to the phagolysosome: promoting breakdown of the corpse membrane within the lumen1. However, how Atg8/LC3 transduces the signal and which factors carry out membrane breakdown remain a mystery. In Aim 3, we tease apart the localization and roles of Atg8/LC3 proteins and their interactors to define the mechanisms of corpse membrane breakdown within the phagolysosome. Membrane breakdown is the limiting factor for the degradation of nucleic acids and proteins within phagolysosomal cargos, which is especially important for MHC class II-expressing immune cells, where lysosomal breakdown products are used for antigen presentation. As defects in LC3-associated phagocytosis have been tied to autoimmune disease in mammals, determining how C. elegans cells promote corpse membrane breakdown can provide new insights into conserved mechanisms in humans during immune signaling, inflammation, and autoimmune disease in addition to pathogen clearance.

非凋亡性细胞程序性死亡和身体清除的机制项目摘要/摘要许多细胞被编程为在发育和动态平衡期间死亡，但并不是所有的细胞都通过细胞凋亡。此外，癌细胞已被证明对促进凋亡的疗法具有抵抗力。因此，它是重要的是要了解细胞死亡的非凋亡机制，以及死亡细胞如何发出清除信号。此外，身体清除和吞噬酶体降解对于避免炎症和自体感染也很重要。免疫疾病，但我们对所有这些过程的理解存在重大差距。我们确定线虫极体是非凋亡性细胞，经历一种未知形式的程序性坏死(Fazeli等人，Cell Rep 2018)1。使用这个遗传模型，我们建议识别在AIM 1中，导致坏死性细胞死亡和膜完整性丧失的机制，最初侧重于非基于一名本科生产生的初步数据的凋亡半胱氨酸酶和扰乱酶调节因子学生。这些有针对性的筛查将确定极体死亡是否与已建立的程序性坏死模式或定义了一种未被发现的细胞死亡类型，这可能提供了洞察力人类在健康和疾病期间的不明细胞死亡模式。我们发现极体外化磷脂酰丝氨酸(PS)，并被胚胎细胞用与LC3相关的吞噬作用。然而，在坏死细胞中调节PS暴露的信号通路是不知道。在一名本科生的初步数据中，我们发现了至少四种多余的脂肪 PS暴露所需的扰乱酶，并通过全部突变来减少PS暴露。这方法是目标2中的几个方法之一，它将允许我们定义调节吞噬的信号通路以及Atg8/Lc3家族蛋白被募集到吞噬小体中。这些信号通路在其他类型的细胞死亡中可能是保守的，包括凋亡，提供了对清理身体的机制。我们还发现了ATG8/LC3招募到吞噬溶酶体的第一个作用：促进分解管腔内的尸膜。然而，Atg8/LC3如何传递信号以及哪些因素进行膜破裂仍然是一个谜。在目标3中，我们梳理了 Atg8/Lc3蛋白及其相互作用蛋白在确定身体膜破裂机制中的作用噬菌体。膜破裂是核酸和蛋白质降解的限制因素在吞噬小体内，这对表达MHC II类的免疫细胞尤其重要，其中溶酶体分解产物用于抗原呈递。Lc3相关吞噬功能的AS缺陷与哺乳动物的自身免疫性疾病有关，这决定了线虫细胞如何促进身体膜破裂可以为人类免疫过程中的保守机制提供新的见解除病原体清除外，信号、炎症和自身免疫性疾病。