一個新穎的DEAD-box蛋白於NLRP3發炎小體中的角色

Abstract

發炎小體(the inflammasomes)為一蛋白質複合體，其功能為將第一型半胱天冬酶(caspase-1)活化並因此催化第一型介白素 β (IL-1β)與第十八型介白素(IL-18)的成熟。發炎小體適度的活化可清除病原體，但失控的發炎反應已被報導和許多癌症及自體炎症性疾病相關。我們實驗室發現了第二型纖溶酶原激活物抑制酶(PAI-2)能在受LPS刺激的巨噬細胞中抑制第一型介白素β的成熟。我們實驗室進行了酵母雙雜交實驗發現一核醣核酸解旋脢(DDX)為PAI-2的結合蛋白。然而，DDX在調控發炎小體的角色仍是未知的。我們研究發現當利用RNA干擾技術(RNA interference)抑制DDX的基因表現並處以細菌內毒素脂多醣體 (LPS，Lipopolysaccharide)後，不管是在人類巨噬細胞THP-1細胞株或是小鼠骨髓細胞分化成的巨噬細胞(BMDM)中都大幅提高成熟型第一型半胱天冬酶活性以及成熟型第一型介白素β的釋放。此外THP-1細胞在處以LPS後DDX會從細胞核轉到細胞質，而DDX的出核對它抑制發炎小體是很重要的。在人類胚腎細胞株HEK293T內證實DDX會和其中一種發炎小體蛋白NLRP3的LRR區域結合。因為LRR區域對於NLRP3跑到粒線體並結合接合蛋白ASC，我們發現NLRP3和ASC的結合會受到DDX存在下的干擾。這些研究結果證實DDX能夠負調控NLRP3發炎小體複合體的形成進而抑制第一型半胱天冬酶活性以及成熟型第一型介白素β的活性。

Inflammasome, a multiple protein complex modulating caspase-1 activity, regulates the proteolytic cleavage of proinflammatory cytokines, IL-1 sand IL-18, into their active forms. Inflammasomes play a crucial role in the clearance of pathogens, however, deregulated inflammasome activity contributes the pathogenesis of various diseases including auto-inflammatory diseases and cancers. Our previous studies showed that PAI-2 is a negative regulator of NLRP3 inflammasome activation. We previously identified a novel DEAD box-containing protein, DDX, using PAI-2 as a bait in a yeast two-hybrid screening. However, the role of DDX in regulation of the inlammasome activation remains unknown. In this research, we found that caspase-1 activation and mature IL-1β s production were largely enhanced upon LPS challenge in DDX-silenced THP-1 macrophages and bone marrow derived macrophages (BMDMs). In addition, DDX migrated from the nucleus to the cytoplasm upon LPS stimulation, which is required for its inhibitory role in NLRP3 inflammasome activation. DDX specifically interacted with the LRR motif of NLRP3 via its DEAD domain. Furthermore, due to the crucial role of the NLRP3 LRR domain in recruitment of NLRP3 to mitochondria and binding to its adaptor ASC, we found that the interaction of NLRP3 and ASC was downregulated in the presence of DDX. Our results suggest that DDX suppresses the formation of the NLRP3 inflammasome complex leading to negative regulation of caspase-1 activation and IL-1β s production.