探討第二型穿膜絲胺酸蛋白酶中之Hepsin對第一型干擾素表現之調控

Abstract

第一型干擾素的誘發和生成對於抗病毒初級免疫反應的形成是非常重要的。當細胞遭受病毒感染時，RIG-1會辨認病毒的核糖核酸(RNA)並活化下游的銜接蛋白質MAVS；cGAS則會辨認細胞質中病原體的去氧核醣核酸(DNA)並活化下游的銜接蛋白質STING。MAVS和STING會接著活化下游的訊息傳導路徑，促使轉錄因子IRF3、IRF7和NF-κB的活化，並進入細胞核誘導第一型干擾素生成。許多病毒的蛋白酶具有切割第一型干擾素生成訊型傳遞路徑中銜接蛋白質的能力，並藉此躲避宿主的免疫反應；例如C型肝炎病毒的蛋白酶NS3/4A可以切割MAVS，使得MAVS離開粒線體並減少第一型干擾素的生成；除此之外，一些人類蛋白酶抑制劑，像是PAI-2和其他絲安酸蛋白酶抑制劑，被發現可以負調控NF-κB訊息傳遞路徑。然而，是否人類絲安酸蛋白酶也參與調控病毒所誘發的第一型干擾素生成，以此維持干擾素的表現，目前尚不清楚。在本篇研究中，我們發現在多種不同的細胞株內，第二型穿膜絲胺酸蛋白酶中之Hepsin可以抑制細胞因感染病毒而誘發的第一型干擾素產生。其中在肝細胞株中可以測得Hepsin的表現，但並非所有細胞株均會表現Hepsin。此外，在Hepsin基因剔除的小鼠胚胎纖維母細胞中可以測得IFNβ有較高的表現。而在Hepsin和MAVS或N-RIG的共轉染細胞中，Hepsin依然可以減少 IFNβ promoter activity；但在Hepsin和TBK-1或IRF3-5D的共轉染細胞中，IFNβ promoter activity則不受到影響。由此可以推斷出，Hepsin的作用位置應在MAVS的下游以及TBK-1的上游。我們也發現只有野生型的Hepsin具有抑制第一型干擾素生成的能力，而蛋白酶活性缺乏的突變型Hepsin則不具有此功能。另外，根據蛋白質在細胞中座落的位置以及Hepsin的推測切割位，我們認為Hepsin是藉由減少STING的蛋白表現量來抑制第一型干擾素的訊息傳遞路徑。最後，我們透過共沈澱免疫法和螢光染色法來確認Hepsin和STING之間確實具有交互作用，並且兩種蛋白質座落在細胞中的位置非常靠近。總而言之，本篇研究的結果發現了Hepsin新功能，認為Hepsin可以在肝細胞中調控第一型干擾素的生成。

The induction of type I interferon (IFN) is critical for antiviral innate immune response. During virus infection, Retinoic acid-inducible gene I (RIG-I) recognizes the viral RNA and activates Mitochondrial antiviral-signaling protein (MAVS). Cyclic GMP-AMP (cGAMP) synthase (cGAS) detects cytosolic pathogenic DNA and activates stimulator of interferon genes (STING). MAVS and STING further trigger downstream signaling to activate interferon regulatory factor 3 (IRF3) or nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) to induce type I IFN. Many viral proteases cleave adapter proteins in the IFN induction pathway, such as HCV NS3/4A cleaving MAVS, as a strategy to evade anti-viral innate immunity. Besides, some serine protease inhibitors, such as PAI-2 and other serine protease inhibitors, could negatively regulate NF-κB pathway. However, it is not clear whether host protease may also regulate virus-induced type I IFN induction pathway to control the steadily low expression of IFN in cells. Here we report that Hepsin (HPN), which belongs to type II transmembrane serine protease family, could inhibit the production of type I IFN during RNA virus infections in several cell-lines. Hepsin expression could be detected in hepatocytes but not ubiquitously in all cell types. Knocking-out Hepsin in mouse embryonic fibroblasts resulted in higher expression of IFNβ. Besides, when co-transfected with MAVS or N-RIG, which is constitutively activating IFNβ expression, Hepsin could decrease IFNβ promoter activity. However, under the expression of TBK1 or IRF3-5D, Hepsin could not reduce IFNβ promoter activity, suggesting that Hepsin might target certain adaptor proteins downstream of MAVS and upstream of TBK1. We showed an inverse correlation with wild-type Hepsin expression but not with the protease-deficient mutant Hepsin in the IFNβ promoter activities. Furthermore, based on the location of protein and putative cleavage site of HPN, it is likely that HPN suppressed IFNβ induction pathway by decreasing STING. Finally, the co-immunoprecipitation and immunofluorescence staining showed that HPN interacted with STING. Consequently, these results reveal a novel role of Hepsin in regulating the type I IFN induction pathway in hepatocytes.