Sušjan Petra, Roškar Samo, Hafner-Bratkovič Iva
Department of Synthetic Biology and Immunology, National Institute of Chemistry, Hajdrihova 19, 1000 Ljubljana, Slovenia; Graduate School of Biomedicine, University of Ljubljana, 1000 Ljubljana, Slovenia.
Uniform Second-Level Master's Program of Medicine, Medical Faculty, University of Ljubljana, Ljubljana 1000, Slovenia.
Biochem Biophys Res Commun. 2017 Feb 5;483(2):823-828. doi: 10.1016/j.bbrc.2017.01.008. Epub 2017 Jan 5.
NLRP3 inflammasome is a multiprotein platform for the activation of caspase-1. Despite the increasing number of reports linking NLRP3 inflammasome to a variety of diseases, the mechanism behind the NLRP3 activation remains elusive, especially in terms of the early stages which are critical to the NLRP3 inflammasome assembly. In the present study we aimed to determine the minimal oligomerization state required for the NLRP3 inflammasome activation. For this purpose, NLRP3 pyrin domain (NLRP3) was fused to various dimerization and trimerization domains. The constructs were expressed under the inducible promoter in mouse macrophages lacking endogenous NLRP3. Dimerization of the NLRP3 either in parallel or in antiparallel orientation was insufficient for the inflammasome activation. Trimerization of the NLRP3 with the foldon domain, however, induced pyroptosis and robust IL-1β maturation, which was caspase-1 dependent. Interestingly, foldon-induced constitutive activation is resistant to inhibition with NLRP3-specific inhibitor MCC950 and does not lead to ASC speck formation. Although we cannot exclude that wild-type NLRP3 forms higher oligomer species similar to NLRP1 or NLRC4, our results clearly demonstrate that efficient IL-1β response can be achieved by the induced trimerization of the NLRP3 domain.
NLRP3炎性小体是一种用于激活半胱天冬酶-1的多蛋白平台。尽管将NLRP3炎性小体与多种疾病联系起来的报道越来越多，但NLRP3激活背后的机制仍然难以捉摸，尤其是在对NLRP3炎性小体组装至关重要的早期阶段。在本研究中，我们旨在确定NLRP3炎性小体激活所需的最小寡聚化状态。为此，将NLRP3吡啉结构域（NLRP3）与各种二聚化和三聚化结构域融合。构建体在缺乏内源性NLRP3的小鼠巨噬细胞中的诱导型启动子下表达。NLRP3以平行或反平行方向二聚化不足以激活炎性小体。然而，NLRP3与折叠结构域三聚化可诱导细胞焦亡和强大的IL-1β成熟，这依赖于半胱天冬酶-1。有趣的是，折叠结构域诱导的组成性激活对NLRP3特异性抑制剂MCC950的抑制具有抗性，并且不会导致ASC斑点形成。虽然我们不能排除野生型NLRP3形成类似于NLRP1或NLRC4的更高寡聚体物种，但我们的结果清楚地表明，通过诱导NLRP3结构域三聚化可以实现有效的IL-1β反应。
