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

项目摘要 坏死性凋亡是最近发现的免疫原性坏死细胞死亡途径。与细胞凋亡不同， 通过细胞肿胀、膜破裂和损伤相关分子模式（DAMP）的释放。它牵涉到 一系列人类疾病，包括感染、炎症、组织损伤、癌症和神经变性。上游 信号，包括细胞因子如TNF、病毒感染或细菌感染会聚于受体相互作用激酶3 （RIPK 3）及其底物混合谱系激酶样蛋白MLKL。RIPK 3对MLKL的磷酸化驱动MLKL 聚合和膜易位，导致质膜破坏。然而，如何MLKL膜 易位是如何调节的以及膜破裂是如何实现的仍在争论中。本提案的总体目标是 确定一种新的膜蛋白MADMAN在发育和疾病状态期间的坏死性凋亡中的作用。使用 通过全基因组CRISPR-Cas9敲除筛选，我们鉴定了一种新的蛋白质，该蛋白质是由 MLKL的N-末端结构域（NTD）的二聚化。我们将这种蛋白质命名为MADMAN，用于MLKL相关膜 坏死性凋亡激活剂MADMAN含有两个假定的跨膜结构域，并形成二硫键连接的 溶酶体膜上的低聚物。我们的初步结果表明，MADMAN与MLKL相互作用， 坏死性凋亡诱导并将MLKL募集至溶酶体膜，导致溶酶体膜透化 （LMP）。此外，MADMAN的过表达足以诱导MLKL依赖性坏死性凋亡。重要的是， Madman-/-小鼠对TNF诱导的全身炎症反应综合征（SIRS）具有抗性，证实了其重要性。 在体内坏死性凋亡中的作用。在本提案中，我们希望定义MADMAN调节MLKL的机制 激活坏死性凋亡。具体来说，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定位。我们还将调查MADMAN如何推广MLKL 聚合，导致LMP。此外，我们将研究Madman-/-小鼠如何对TNF诱导的系统性免疫应答。 炎症反应综合征和如果Madman的丧失挽救了由半胱天冬酶8缺陷引起的胚胎致死。最后， 我们将研究LMP和溶酶体蛋白酶在坏死性凋亡执行中的作用。回答这些问题将导致 我们更好地了解如何在分子水平上执行坏死性凋亡，并提供新的治疗策略， 与坏死性凋亡过度活化有关的疾病。我们的方案结合了正向遗传学，反向遗传学 遗传学，生物化学和细胞生物学方法来破译一种新的膜蛋白MADMAN在 坏死性凋亡它将回答关于MLKL膜易位、聚合和 膜破裂这些研究将为坏死性凋亡途径提供新的分子见解，这可能导致 用于治疗坏死性凋亡相关的炎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.