Deconstructing the Checkpoints of Necroptosis

长期以来，细胞凋亡被认为是发育、稳态和疾病过程中细胞程序性死亡的唯一形式，而坏死则被认为是一个不受调控和无法控制的过程。现在有证据表明，坏死也可以以一种受监管的方式发生。程序性坏死，即‘坏死下垂’，在宿主-病原体相互作用中起着至关重要的作用，在宿主-防御机制中被触发，以消除病原体感染的细胞。然而，坏死性上睑下垂也参与了包括缺血性损伤、神经变性和病毒感染在内的疾病的发病机制。此外，坏死性下垂还引起了癌症研究人员的极大兴趣，因为它有可能克服肿瘤对细胞凋亡的抵抗力，而且它比细胞凋亡更具免疫原性，使肿瘤成为免疫攻击的标志。由于这些原因，人们非常有兴趣更好地了解坏死性下垂是如何被激活的，以及这一潜在的灾难性事件是如何被调控的。坏死性上睑下垂是由MLKL介导的，MLKL是一种膜通透性假激酶，在激活后转移到质膜上。虽然坏死性下垂的信号通路因其潜在的治疗潜力而引起了人们的极大关注，但人们对坏死性下垂的调控机制以及MLKL如何转移到质膜上的热点引发坏死性下垂知之甚少。我们现在已经发现泛素(Ub)信号系统对坏死性下垂起着关键的调节作用，MLKL的泛素化是MLKL运输到质膜所必需的。此外，我们还鉴定了几个可能作为决定坏死链检查点的MLKL调节Ub-E3连接酶和去泛素化酶。目的：本研究的目的是确定活性MLKL移位到质膜并聚集在热点处导致溶细胞死亡的机制。我们还将确定MLKL积聚的细胞间结构，并表征它们在坏死性下垂信号和膜破裂中的作用。为了实现这一点，我们将表征Ub信号系统的分子角色(E3连接酶、去泛素化酶和Ub受体)，这些分子支持MLKL泛素化、运输和在细胞间接触部位的积聚。方法：利用生化、单细胞成像和体内实验的方法，我们将阐明MLKL是如何被E3连接酶泛素化的，去泛素化酶是如何编辑MLKL的泛素化状态的，以及Ub结合蛋白(Ub受体)是如何检测这些信号链的，从而将活性MLKL运送到质膜上的细胞间热点。此外，我们还将揭示已识别的Ub-E3连接酶、去泛素化酶和Ub受体在调节抗病毒宿主防御中的作用。此外，我们将评估桥粒和Flotillins在坏死性下垂信号中的作用。如何利用这些结果更好地理解坏死性下垂信号将引起基础科学家和临床研究人员的极大兴趣，因为它将为未来旨在增强抗病毒防御、抗击癌症和抑制炎症性疾病的治疗策略的设计奠定基础。

For a long time, apoptosis was considered the sole form of programmed cell death during development, homeostasis, and disease, whereas necrosis was regarded as an unregulated and uncontrollable process. Evidence now reveals that necrosis can also occur in a regulated manner. Programmed necrosis, 'necroptosis' plays vital roles during host-pathogen interactions where it is triggered as host-defence mechanism for the elimination of pathogen-infected cells. However, necroptosis also participates in the pathogenesis of diseases, including ischaemic injury, neuro-degeneration, and viral infection. Moreover, necroptosis has also sparked considerable interest among cancer researchers for its potential to overcome tumour resistance to apoptosis, and because it is more immunogenic than apoptosis, flagging up tumours for immunological attack. For these reasons, there has been much interest in obtaining a better understanding of how necroptosis is activated and how this potentially catastrophic event is regulated. Necroptosis is mediated by MLKL, a membrane permeabilizing pseudo-kinase that translocates to the plasma membrane upon its activation. While necroptosis signalling has attracted much attention for its therapeutic potential, little is known how necroptosis is regulated, and how MLKL translocates to hotspots at the plasma membrane to trigger necroptosis.We now have identified that the Ubiquitin (Ub)-signalling system critically regulates necroptosis, and that ubiquitylation of MLKL is required for MLKL to traffic to the plasma membrane. Moreover, we have identified several putative MLKL-regulatory Ub-E3 ligases and deubiquitylating enzymes that might operate as decisive necroptotic checkpoint. Aim:The aim of this proposal is to identify the mechanism through which active MLKL translocates to the plasma membrane where it accumulates at hotspots to cause lytic cell death. We will also identify the intercellular structures at which MLKL accumulates and characterise their contribution to necroptosis signalling and membrane rupture.To achieve this, we will characterise the molecular players of the Ub signalling system (E3 ligases, deubiquitylating enzymes and Ub-receptors) that underpin MLKL ubiquitylation, trafficking and accumulation at intercellular contact sites. Moreover, we will study whether the identified E3 ligases, deubiquitylating enzymes and Ub-receptors contribute to antiviral host defence.Methods:Using biochemical, single-cell imaging and in vivo approaches, we will elucidate how MLKL is ubiquitylated by E3 ligases, how the ubiquitylation status of MLKL is edited by deubiquitylating enzymes, and how such signalling chains are detected by Ub-binding proteins (Ub-receptors) to shuttle active MLKL to intercellular hotspots at the plasma membrane. Moreover, we will unravel the role of the identified Ub-E3 ligases, deubiquitylating enzymes and Ub-receptors in modulating anti-viral host defences. Further, we will evaluate the contribution of desmosomes and Flotillins in necroptosis signalling.How the results will be usedA better understanding of necroptosis signalling will be of enormous interest to basic scientists as well as clinical researchers because it will lay the foundation for the design of future therapeutic strategies aimed at boosting antiviral defence, fighting cancer and suppressing inflammatory diseases.