Deconstructing the Checkpoints of Necroptosis

长期以来，细胞凋亡被认为是发育、稳态和疾病过程中程序性细胞死亡的唯一形式，而坏死被认为是一种不受调节和不可控的过程。现在有证据表明，坏死也可以以调节的方式发生。程序性坏死，“坏死性凋亡”在宿主-病原体相互作用期间起着至关重要的作用，在宿主-病原体相互作用中，它被触发作为消除病原体感染细胞的宿主防御机制。然而，坏死性凋亡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，坏死性凋亡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凋亡的抵抗力，并且因为它比凋亡更具免疫原性，标志着肿瘤的免疫攻击。由于这些原因，人们对更好地了解坏死性凋亡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。坏死性凋亡是由MLKL介导的，MLKL是一种膜透化假激酶，在其活化后易位至质膜。虽然necroptosis信号已经吸引了很多注意力，其治疗潜力，很少有人知道necroptosis是如何调节的，以及MLKL如何易位到热点在质膜上，以触发necroptosis.We现在已经确定，泛素（Ub）信号系统的关键调节necroptosis，和MLKL的泛素化所需的MLKL运输到质膜。此外，我们已经确定了几个假定的MLKL调节Ub-E3连接酶和去泛素化酶，可能作为决定性的坏死性检查点。目的：本提案的目的是确定活性MLKL易位至质膜的机制，MLKL在质膜的热点处积聚，导致溶解性细胞死亡。我们还将确定MLKL积累的细胞间结构，并研究它们对坏死性凋亡信号传导和膜破裂的作用。为了实现这一目标，我们将研究Ub信号传导系统（E3连接酶，去泛素化酶和Ub受体）的分子参与者，这些分子参与者支持MLKL在细胞间接触位点的泛素化，运输和积累。此外，我们将研究是否确定的E3连接酶，deubiquitylating酶和UB-受体有助于抗病毒宿主defensity.Methods：使用生物化学，单细胞成像和在体内的方法，我们将阐明如何MLKL是泛素化的E3连接酶，如何MLKL的泛素化状态编辑deubiquitylating酶，以及如何检测到这样的信号链由UB-结合蛋白（UB-受体）穿梭活性MLKL细胞间的热点质膜。此外，我们将解开的Ub-E3连接酶，去泛素化酶和Ub受体在调节抗病毒宿主防御的作用。此外，我们将评估桥粒和Flotillins在坏死性凋亡信号传导中的贡献。如何使用结果更好地了解坏死性凋亡信号传导将对基础科学家和临床研究人员产生巨大的兴趣，因为它将为设计未来的治疗策略奠定基础，旨在增强抗病毒防御，对抗癌症和抑制炎症性疾病。

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.