The lysosomal Rag-Ragulator complex licenses RIPK1-and caspase-8-mediated pyroptosis by Yersinia
The lysosomal Rag-Ragulator complex licenses RIPK1-and caspase-8-mediated pyroptosis by Yersinia
复制标题
溶酶体 Rag-Ragulator 复合物许可耶尔森氏菌介导 RIPK1 和 caspase-8 介导的细胞焦亡
DOI：
10.1126/science.abg0269
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发表时间：
2021-06-25
期刊：
影响因子：
56.9
通讯作者：
Liu, Xing
中科院分区：
文献类型：
--
作者：
Zheng, Zengzhang;Deng, Wanyan;Liu, Xing
炎症性先天免疫反应是宿主抵御入侵病原体的第一道防线。炎症将免疫细胞招募到感染部位，并激活保护性适应性免疫反应。入侵细菌已经进化出多种方式来干扰宿主的先天性免疫信号，以促进感染。耶尔森尼亚效应蛋白YopJ通过抑制转化生长因子-β激活蛋白1和核因子κB(NF-κB)的激活来抑制促炎细胞因子的产生。为了对抗这种毒力因子，当TAK1被抑制时，宿主细胞启动受体相互作用的丝氨酸-苏氨酸蛋白激酶1(RIPK1)依赖的caspase-8指导的Gasdermin D(GSDMD)裂解和炎性细胞死亡(松下症)。然而，在耶尔森尼病毒感染过程中，RIPK1-caspase-8-GSDMD轴是如何被指示的尚不清楚。病原体刺激炎症细胞死亡的最好研究机制涉及触发胞液传感器，称为炎症小体，它激活炎症caspase(1/4/5/11)来处理促炎细胞因子并导致上睑下垂。GSDMD的炎性半胱氨酸氨基转移酶裂解导致细胞膜毛孔，从而调节下垂和促炎性细胞因子的分泌。当致病过程中分泌的YopJ毒力因子阻断TAK1的激活时，由RIPK1和caspase-8的激活介导的另一条焦链途径被触发。为了确定依赖RIPK1-caspase-8的焦虑症的耶尔辛尼激活的分子机制，我们进行了全基因组CRISPR筛查，使用表达Cas9的永生化小鼠骨髓来源的巨噬细胞感染全基因组文库的单引导RNA编码的慢病毒。对caspase-8或caspase-11依赖的上睑下垂抗性细胞的基因组进行测序，以确定caspase-8或caspase-11依赖的上睑下垂所需的敲除基因。结果筛选发现，溶酶体膜-锚定毛囊蛋白(Flcn)-毛囊相互作用蛋白2(Fnip2)-Rag-Ragator复合体中有多个基因是caspase-8-而不是caspase-11介导的上睑下垂所必需的。Rag-Ragiller超复合体基因的缺失使细胞对TAK1抑制触发的下垂具有高度的抵抗力，这表明溶酶体膜系Rag-Ragator超复合体在依赖RIPK1的caspase-8导向的下垂中发挥了关键和意想不到的作用。根据病原菌的致病机理，将含有Fas相关死亡结构域(FADD)-RIPK1-caspase-8的Fas相关死亡结构域(FADD)-RIPK1-caspase-8复合体引入溶酶体膜栓系Rag-Ragator。RIPK1磷酸化、caspase-8激活和热下垂的激活依赖于Rag鸟苷三磷酸酶(GTPase)活性和Rag-Ragator溶酶体结合，但与雷帕霉素复合体1(MTORC1)的作用靶点无关。相反，Rag-Ragator不调节典型或非典型炎症性下垂。结论本研究揭示了代谢信号在TAK1抑制引起的下垂的指导作用。RAG-Ragator是众所周知的细胞对养分供应和新陈代谢变化反应的关键调节因子。在这里，Rag-Ragator作为一个平台，激活Toll样受体(TLR)或肿瘤坏死因子受体(TNFR)连接后形成的FADD-RIPK1-caspase-8复合体。RAG GTP酶活性是触发该途径的关键。这里找到的角色是…
INTRODUCTIONAn inflammatory innate immune response is a first line of host defense against invading pathogens. Inflammation recruits immune cells to the infection site and activates protective adaptive immune responses. Invasive bacteria have evolved multiple ways to interfere with host innate immune signaling to facilitate infection. TheYersiniaeffector protein YopJ suppresses proinflammatory cytokine production by inhibiting transforming growth factor–β–activated kinase 1 (TAK1) and nuclear factor κB (NF-κB) activation. To counteract this virulence factor, host cells initiate receptor-interacting serine-threonine protein kinase 1 (RIPK1)–dependent caspase-8–directed gasdermin D (GSDMD) cleavage and inflammatory cell death (pyroptosis) when TAK1 is inhibited. However, how the RIPK1–caspase-8–GSDMD axis is instructed duringYersiniainfection is unclear.RATIONALEThe best-studied mechanism by which pathogens stimulate inflammatory cell death involves triggering cytosolic sensors, called inflammasomes, which activate inflammatory caspases (1/4/5/11) to process proinflammatory cytokines and cause pyroptosis. Inflammatory caspase cleavage of GSDMD causes cell membrane pores that mediate both pyroptosis and proinflammatory cytokine secretion. An alternate pyroptotic pathway, mediated by activation of RIPK1 and caspase-8, is triggered when the YopJ virulence factor secreted during pathogenicYersiniainfection blocks TAK1 activation. To determine the molecular mechanisms underlyingYersiniaactivation of RIPK1–caspase-8–dependent pyroptosis, we performed a genome-wide CRISPR screen using Cas9-expressing immortalized mouse bone marrow–derived macrophages infected with a genome-wide library of single-guide RNA–encoding lentiviruses. The genomes of cells resistant to caspase-8– or caspase-11–dependent pyroptosis were sequenced to identify the knocked-out genes required for pyroptosis.RESULTSThe screen identified multiple genes in the lysosomal membrane–anchored Folliculin (Flcn)–Folliculin-interacting protein 2 (Fnip2)–Rag-Ragulator complex as necessary for caspase-8– but not caspase-11–mediated pyroptosis. Deficiency of Rag-Ragulator complex genes rendered cells highly resistant to TAK1 inhibition–triggered pyroptosis, indicating a critical and unexpected role of the lysosomal membrane–tethered Rag-Ragulator supercomplex in RIPK1-dependent caspase-8–directed pyroptosis. In response to pathogenicYersiniaor its mimic [lipopolysaccharide (LPS) plus TAK1 inhibitor], a Fas-associated death domain (FADD)–RIPK1–caspase-8–containing complex was recruited to lysosomal membrane–tethered Rag-Ragulator. Activation of RIPK1 phosphorylation, caspase-8 activation, and pyroptosis depended on Rag guanosine triphosphatase (GTPase) activity and Rag-Ragulator lysosomal binding but was independent of the mechanistic target of rapamycin complex 1 (mTORC1), a well-known Rag-Ragulator–regulated complex. By contrast, Rag-Ragulator did not regulate canonical or noncanonical inflammasome-triggered pyroptosis.CONCLUSIONOur study revealed an instructive role of metabolic signaling in directing TAK1 inhibition–induced pyroptosis during a pathogenic bacterial infection. Rag-Ragulator is a well-known critical regulator of cellular responses to changes in nutrient availability and metabolism. Here, Rag-Ragulator served as a platform for activating a FADD–RIPK1–caspase-8 complex formed in response to Toll-like receptor (TLR) or tumor necrosis factor receptor (TNFR) ligation. Rag GTPase activity was critical for triggering the pathway. The role found here …