一個新穎的核醣核酸解旋酶於發炎反應中的角色

Abstract

近幾年，發炎體(the inflammasomes)這種新興蛋白質複合體在免疫學界中迅速竄起，被證實其是大多數多細胞生物體內用以對抗病原體的一環。發炎體主要牽涉到先天免疫中發炎反應啟動的過程，其具有將第一型半胱天冬酶(caspase-1)活化並間接催化第一型介白素β (IL-1β)與第十八型介白素(IL-18)熟成的能力，以協助宿主對抗病原體。但發炎體究竟是如何被調控的至今尚未明瞭。 過去研究中，指出第二型纖溶酶原激活物抑制酶(PAI-2)在受LPS刺激的巨噬細胞中能夠抑制第一型介白素β的熟成，同時能減少刺激後Annexin V的訊號強度，但第二型纖溶酶原激活物抑制酶是如何做到這點的目前並不清楚。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為第二型纖溶酶原激活物抑制酶的結合蛋白。本篇研究的目標即為透過深入研究兩者關係來進一步瞭解第二型纖溶酶原激活物抑制酶是如何調節第一型介白素β的熟成。 此次研究中，我們在人類胚腎細胞株HEK293T內證實該核醣核酸解旋脢是第二型纖溶酶原激活物抑制酶的結合蛋白。當我們進一步利用RNA干擾技術(RNA interference)抑制該核醣核酸解旋脢的基因表現後，不管是在以分化的人類巨噬細胞THP-1細胞株或是小鼠骨髓細胞分化而成的巨噬細胞(BMDM)中都能大幅提高成熟型第一型半胱天冬酶(mature caspase-1) 活性以及成熟型第一型介白素β (mature IL-1β)的釋放，卻不影響上游前第一型介白素β核醣核酸 (pro-IL-1β mRNA)以及前第一型介白素β蛋白質(pro-IL-1β)的數量，這指出該核醣核酸解旋脢可能涉入發炎體將第一型介白素從前型轉為成熟型的熟成過程。反之，當在HEK293T細胞過度表達該核醣核酸解旋脢時可以抑制重組NLRP3發炎體將前第一型介白素β轉化為成熟型的效率，證實該核醣核酸解旋脢能夠負調控發炎體的活性。

Innate immunity is an evolutionarily conserved defense mechanism against infection. Upon infection, the innate immune response is immediately initiated to clear pathogens and repair damage tissues. Recently, a novel system named the inflammasome, a multiple protein complexes modulating caspase-1 activity, has been described. Activation of the inflammasome results in the cleavage of precursors of pro-inflammatory cytokines IL-1β and IL-18, into their bioactive forms. The inflammasome has been shown to play a crucial role in host defense against bacterial and viral infection in vivo. A growing body of evidence, however, demonstrated that dysregulation of the inflammasome activity has been associated with a variety of human diseases including auto-inflammatory and metabolic diseases. Thus, activation of the inflammasome must be tightly controlled, but its positive and negative regulation remains largely unknown. Previous studies demonstrated that the overexpression of PAI-2, a transcriptional target gene of NF-κB and p38, suppressed IL-1β production is still unknown. Our lab recently screened PAI-2-interacting proteins using the yeast two-hybrid system and identified several candidate proteins. Amongst these proteins, one of which is one member of DEAD-box protein family. DEAD-box protein family including RIG-I, MDA5, and DDX41 which contribute to innate immune responses against viral infection. This protein family is known for its abilities in RNA metabolism and RNA-protein crosstalk; yet, the exact function of the DEAD-box protein we screened has not been proposed to date. Thus, we aim to study the role of this DEAD-box protein in LPS-induced IL-1β production. In this study, we found that ablation of this DEAD-box protein in differentiated THP-1 macrophages and bone marrow derived macrophages (BMDMs) largely enhance caspase-1 activation and mature IL-1β production upon LPS challenge. Besides, in response to LPS treatment, no significant alteration on pro-IL-1β expression was observed in this DEAD-box protein-ablated cells compared to wild type cells. Overexpression of this DEAD-box protein suppressed mature IL-1β production in the reconstitution of NLRP3 inflammasome in HEK293T cells. Our results strongly suggest that this DEAD-box protein may act as a negative regulator of Inflammasome activation.