来自美国基因泰克公司的研究人员发现应答细菌感染时NOD样受体NLRC4的磷酸化对炎症小体激活至关重要。

-2012年10月25日《自然》


中文翻译


【题目】NLRC4磷酸化对炎症小体激活至关重要

【译文】NLRC4是NOD样受体家族胞质成员之一,在固有免疫细胞中表达。它可以间接感受细菌鞭毛蛋白和III型分泌系统,并通过组装一种可促进半胱天冬酶1活化和细胞程序性死亡的炎症小体复合物而产生应答。本研究利用表达羧基端带有3×Flag 标记的NLRC4的基因敲入小鼠,以鉴定NLRC4在单一、进化保守的残基Ser 533的磷酸化,然后用鼠伤害沙门氏菌感染巨噬细胞。利用NLRC4磷酸化的Ser 533抗体的蛋白印迹证实这种转录后修饰作用仅发生在已知应答NLRC4的刺激物存在的情况下,而非相关蛋白NLRP3 或AIM2。Nlrc4−/−巨噬细胞利用NLRC4突变体S533A重组,与那些利用野生型NLRC4重组的不同,这种巨噬细胞应答沙门氏菌时不会激活半胱天冬酶1和细胞程序性死亡,表明S533磷酸化对于NLRC4炎症小体功能至关重要。相反,在没有感染的情况下,蛋白磷酸化的NLRC4 S533D会导致巨噬细胞快速程序性死亡。生化纯化NLRC4磷酸化酶和一些激酶抑制剂证实PRKCD (PKCδ)是一种候选的NLRC4激酶。体外重组PKCδ磷酸化的NLRC4 S533,巨噬细胞溶菌产物中PKCδ的免疫耗竭阻断了体外NLRC4 S533的磷酸化,而且Prkcd−/−巨噬细胞强烈抑制半胱天冬酶1的活化和IL-1β的分泌,尤其是在应答沙门氏菌感染时。在鼠伤害沙门氏菌感染期间,磷酸化缺陷的NLRC4 S533A则不能招募半胱天冬酶1前体,也不能组装炎症小体复合物,因此NLRC4 S533的磷酸化可能驱动对NLRC4炎症小体活性和宿主固有免疫必需的构象变化。

英文原稿


[Title]: Phosphorylation of NLRC4 is critical for inflammasome activation

 [Authors]:Yan Qu,1, 10 Shahram Misaghi,2, 10 Anita Izrael-Tomasevic,3 Kim Newton,1 Laurie L. Gilmour,4 Mohamed Lamkanfi,5, 6 Salina Louie,2 Nobuhiko Kayagaki,1 Jinfeng Liu,7 László Kömüves,8 James E. Cupp,4 David Arnott,3 Denise Monack9 & Vishva M. Dixit1

[Abstract]NLRC4 is a cytosolic member of the NOD-like receptor family that is expressed in innate immune cells. It senses indirectly bacterial flagellin and type III secretion systems, and responds by assembling an inflammasome complex that promotes caspase-1 activation and pyroptosis. Here we use knock-in mice expressing NLRC4 with a carboxy-terminal 3×Flag tag to identify phosphorylation of NLRC4 on a single, evolutionarily conserved residue, Ser 533, following infection of macrophages with Salmonella enterica serovar Typhimurium (also known as Salmonella typhimurium). Western blotting with a NLRC4 phospho-Ser 533 antibody confirmed that this post-translational modification occurs only in the presence of stimuli known to engage NLRC4 and not the related protein NLRP3 or AIM2. Nlrc4−/− macrophages reconstituted with NLRC4 mutant S533A, unlike those reconstituted with wild-type NLRC4, did not activate caspase-1 and pyroptosis in response to S. typhimurium, indicating that S533 phosphorylation is critical for NLRC4 inflammasome function. Conversely, phosphomimetic NLRC4 S533D caused rapid macrophage pyroptosis without infection. Biochemical purification of the NLRC4-phosphorylating activity and a screen of kinase inhibitors identified PRKCD (PKCδ) as a candidate NLRC4 kinase. Recombinant PKCδ phosphorylated NLRC4 S533 in vitro, immunodepletion of PKCδ from macrophage lysates blocked NLRC4 S533 phosphorylation in vitro, and Prkcd−/− macrophages exhibited greatly attenuated caspase-1 activation and IL-1β secretion specifically in response to S. typhimurium. Phosphorylation-defective NLRC4 S533A failed to recruit procaspase-1 and did not assemble inflammasome specks during S. typhimurium infection, so phosphorylation of NLRC4 S533 probably drives conformational changes necessary for NLRC4 inflammasome activity and host innate immunity.

原文地址

http://www.nature.com/nature/journal/v490/n7421/full/nature11429.html

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