Regulation of cell death and disease by a novel membrane protein MADMAN.

项目总结 坏死性下垂是近年来发现的一种免疫性坏死性细胞死亡途径。与细胞凋亡不同，它的特征是 通过细胞肿胀、膜破裂和释放受损的相关分子模式(DAMP)。它被牵连到 一系列人类疾病，包括感染、炎症、组织损伤、癌症和神经退化。上游 信号，包括细胞因子，如肿瘤坏死因子、病毒感染或细菌感染，汇聚在受体相互作用的激酶3上。 (RIPK3)与其底物混合的谱系蛋白样蛋白MLKL。RIPK3对MLKL的磷酸化驱动MLKL 聚合和膜移位，导致质膜破坏。然而，MLKL膜如何 转位是受调控的，膜破裂是如何实现的仍在争论中。这项提案的总体目标是 明确一种新的膜蛋白Madman在坏死性下垂发育和疾病状态中的作用。使用 全基因组CRISPR-Cas9基因敲除筛选，我们鉴定了一种新的蛋白质，它是由 MLKL N-末端结构域(NTD)的二聚化。我们将该蛋白命名为Madman for MLKL相关膜 坏死性下垂的激活剂。Madman含有两个假定的跨膜结构域，形成二硫键连接的结构域 溶酶体膜上的低聚物。我们的初步结果表明Madman与MLKL在 诱导坏死性下垂并将MLKL招募到溶酶体膜，导致溶酶体膜通透性 (LMP)。此外，MADMAN的过度表达足以诱导MLKL依赖的坏死性下垂。重要的是 Madman-/-小鼠对肿瘤坏死因子诱导的全身炎症反应综合征(SIRS)具有抵抗力，证实了其重要性 在体内的坏死性上睑下垂中的作用。在这项提案中，我们希望定义Madman监管MLKL的机制 激活以激活坏死性下垂。具体地说，我们将描述Madman的膜定位，并定义如何 Madman和MLKL相互作用调节MLKL的定位。我们还将调查Madman如何推广MLKL 导致LMP的聚合。此外，我们将研究Madman-/-小鼠对肿瘤坏死因子诱导的全身性 炎性反应综合征和如果失去Madman可以挽救因Caspase 8缺乏而导致的胚胎死亡。最后， 我们将研究LMP和溶酶体蛋白在坏死性下垂执行中的作用。回答这些问题将导致 使我们更好地了解坏死性下垂是如何在分子水平上执行的，并为以下疾病提供新的治疗策略 与坏死性下垂过度活动相关的疾病。我们的方案结合了正向遗传学、反向遗传学 遗传学、生化和细胞生物学方法破译一种新的膜蛋白狂人在 坏死性下垂。它将回答关于MLKL膜转位，聚合和 膜破裂。这些研究将提供对坏死性下垂途径的新的分子见解，这可能导致 治疗坏死性下垂相关炎症性和感染性疾病的新治疗策略。

Project summary Necroptosis is a recently discovered immunogenic necrotic cell death pathway. Distinct from apoptosis, it is characterized by cellular swelling, membrane rupture and release of damaged-associated molecular patterns (DAMPs). It is implicated in an array of human diseases, including infection, inflammation, tissue injury, cancer and neurodegeneration. Upstream signals, including cytokines such as TNF, viral infection or bacterial infection converge on receptor-interacting kinase 3 (RIPK3) and its substrate mixed lineage kinase-like protein MLKL. Phosphorylation of MLKL by RIPK3 drives MLKL polymerization and membrane translocation, leading to plasma membrane disruption. However, how MLKL membrane translocation is regulated and how membrane rupture is achieved are still under debate. The overall goal of this proposal is to define the role of a novel membrane protein MADMAN in necroptosis during development and disease state. Using a whole-genome CRISPR-Cas9 knockout screen, we identified a novel protein that is required for necroptosis induced by dimerization of N-terminal domain (NTD) of MLKL. We named this protein MADMAN for MLKL-associated membrane activator of necroptosis. MADMAN contains two putative transmembrane domains and forms disulfide bond-linked oligomers on the lysosomal membrane. Our preliminary results demonstrate that MADMAN interacts with MLKL upon necroptosis induction and recruits MLKL to the lysosomal membrane, leading to lysosomal membrane permeabilization (LMP). Furthermore, overexpression of MADMAN is sufficient to induce MLKL-dependent necroptosis. Importantly, Madman-/- mice are resistant to TNF-induced systemic inflammatory response syndrome (SIRS), confirming its essential function in necroptosis in vivo. In this proposal, we want to define the mechanism by which MADMAN regulates MLKL activation to activate necroptosis. Specifically, we will characterize membrane localization of MADMAN and define how MADMAN and MLKL interaction regulates MLKL localization. We will also investigate how MADMAN promotes MLKL polymerization which leads to LMP. Furthermore, we will examine how Madman-/- mice respond to TNF-induced systemic inflammatory response syndrome and if loss of Madman rescues embryonic lethality caused by Caspase 8 deficiency. Lastly, we will investigate the role of LMP and lysosomal proteases in necroptosis execution. Answering these questions will lead us to a better understanding of how necroptosis is executed at the molecular level and provide new therapeutic strategies for diseases associated with hyperactivation of necroptosis. Our proposal uses a combination of forward genetics, reverse genetics, biochemical and cell biology approaches to decipher the role of a novel membrane protein MADMAN in necroptosis. It will answer very important questions regarding MLKL membrane translocation, polymerization and membrane disruption. These studies will provide novel molecular insights into the necroptosis pathway, which may lead to new therapeutic strategies for treating necroptosis-associated inflammatory and infectious disease.