Deconstructing the Checkpoints of Necroptosis

长期以来，细胞凋亡被认为是细胞在发育、体内平衡和疾病过程中程序性死亡的唯一形式，而坏死则被认为是一个不受调节和不可控的过程。现在有证据表明，坏死也可以以一种受调节的方式发生。程序性坏死在宿主-病原体相互作用中起着至关重要的作用，它作为宿主防御机制被触发，以消除病原体感染的细胞。然而，坏死性上睑下垂也参与疾病的发病机制，包括缺血性损伤、神经变性和病毒感染。此外，坏死下垂也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，并且因为它比细胞凋亡更具免疫原性，标记肿瘤进行免疫攻击。由于这些原因，人们对更好地了解坏死性下垂是如何被激活的以及这种潜在的灾难性事件是如何被调节的非常感兴趣。坏死下垂是由MLKL介导的，MLKL是一种膜渗透性伪激酶，激活后易位到质膜上。虽然坏死下垂信号因其治疗潜力而备受关注，但人们对坏死下垂是如何调节的，以及MLKL是如何易位到质膜热点触发坏死下垂的却知之甚少。我们现在已经确定了泛素（Ub）信号系统对坏死坏死有关键的调节作用，并且MLKL的泛素化是MLKL转运到质膜所必需的。此外，我们已经确定了几种可能的mlkl调节的Ub-E3连接酶和去泛素化酶，它们可能作为决定性的坏死性坏死检查点起作用。目的：本研究的目的是确定活性MLKL易位到质膜的机制，并在质膜的热点处积累，从而导致溶解性细胞死亡。我们还将确定MLKL积聚的细胞间结构，并描述它们对坏死坏死信号传导和膜破裂的贡献。为了实现这一目标，我们将描述Ub信号系统的分子参与者（E3连接酶、去泛素化酶和Ub受体），它们支持MLKL泛素化、细胞间接触位点的运输和积累。此外，我们将研究所鉴定的E3连接酶、去泛素化酶和ub受体是否有助于抗病毒宿主防御。方法：利用生物化学、单细胞成像和体内方法，我们将阐明E3连接酶如何使MLKL泛素化，去泛素化酶如何编辑MLKL的泛素化状态，以及ub结合蛋白（ub受体）如何检测这些信号链，将活性MLKL输送到质膜上的细胞间热点。此外，我们将揭示已鉴定的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在坏死坏死信号传导中的作用。更好地理解坏死坏死信号将引起基础科学家和临床研究人员的极大兴趣，因为它将为设计未来旨在增强抗病毒防御、对抗癌症和抑制炎症性疾病的治疗策略奠定基础。

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.